The PM₁₀ extract (10 ng/L and 10 μ g/L) was found to inhibited A549 cell proliferation in a time-dependent manner (Figure 1). In addition, the chemical parameters did not inhibit cell proliferation. Next, extracellular LDH enzyme activity was determined to further assess the number of membrane-damaged cells. After 12 hours the LDH level released from A549 cells was observed significantly increasing in both of PM10 exposed groups compared with that from control cells (Figure 2).

图1

Fig. 1

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图1 暴露于PM10 溶液对 A549 细胞增殖的影响 使用 WST-8的比色测定法确定对细胞增殖的抑制作用, 并通过比较在指定时间点暴露与未暴露于 PM10的细胞培养基的吸收值来计算。 与阴性对照组比较, * P< 0.05。Fig. 1 Effect of PM10 extract on A549 cell proliferation The inhibitory effect on cell proliferation was determined using the WST-8 colorimetric assay and calculated by comparing the absorption of the culture medium of PM10-exposed cells with that of non-PM10-exposed cells at the indicated time points. * P< 0.05 compared with the control.

图2

Fig. 2

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	图2 暴露于 PM10溶液后 A549 细胞释放 LDH 通过比较暴露与未暴露于 PM10 的细胞培养基的吸收来计算 LDH 释放。与阴性对照组比较, * P< 0.05。Fig. 2 Release of LDH from A549 cells after exposure to PM10 water extract LDH release was calculated by comparing the absorption of culture medium of PM-exposed cells with that of non-PM-exposed cells. * P< 0.05 compared with the control.

In this study, we found that PM₁₀ significantly inhibits the proliferation of alveolar epithelial cells in a time- and concentration-dependent manner. These findings are consistent with those of previous studies, which demonstrated that cell proliferation significantly decreases in a concentration-dependent manner in PM_2.5-treated A549 cells^{[9, 10, 11]}. Nonetheless, the two PM₁₀ concentrations tested (10 ng/L and 10 μ g/L) did not exert significant cytotoxic effects^[12], which contrasted with the result of previous studies, which showed a significant increase in LDH release in a concentration-dependent manner in PM_2.5-treated A549 cells^{[10, 11]}. These different effects of PM on cells may be explained by the low concentrations used in the present study. As shown in Figure 1, the Pvalue of the cell viability data under 10 ng/L PM₁₀ treatment for 0 and 12 h with that of non-PM₁₀-exposed cells at the indicated time points was low. Therefore, it can be considered that there is significant difference in cell viability at 10 ng/L. We used the 10 µ g/L treatment to compare with higher concentrations, but no obvious difference in cell activity at higher concentration was found. The results in this study indicate that the effects of PM₁₀ depended on time, and its relationship with concentration was not significant. In this study, 10 ng/L was the experimental group, and higher concentration of 10 µ g/L was set as an experimental comparison.

Numerous studies have experimentally used relatively high concentrations of PM₁₀; however, the PM₁₀ concentration is low in the atmosphere, and the amount of PM₁₀ that can enter the alveoli is very small compared to the concentration dependence^{[10, 11]}. Only a few studies have performed experiments with such low concentrations (10 ng/L), which are lower than the standard PM₁₀ concentrations in domestic and foreign haze environments (100 μ g/L). Indeed, cell morphology and release of LDH are unaffected after 24 h of exposure to 125 and 250 μ g/L PM^[13].

Oxidative stress is often considered to be a primary effect of PM toxicity. PM-induced reactive oxygen species (ROS) formation has been widely reported and is associated with metals and organic species within particles^[14]. In addition to the well-known role of some metals in the generation of ROS, the implications of organic compounds were recently confirmed^[15]. However, further experiments are necessary to confirm these findings.

Measurement of cell proliferation and viability has become a core investigational approach in the field of life sciences^[16]. The demand for sensitive, reliable, rapid, and simple methods has led to the development of various detection methods, including the tetrazolium salt (WST)-based assay. Notably, unlike 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), which splits into water-insoluble formazan crystals and must be dissolved after cleavage, WST produces water-soluble cleavage products: 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and 3-(4, 5-dimethylthiazol-20yl)-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS), that can be measured without an additional solubilization steps. Furthermore, WST is more stable than XTT and MTS^[17].

Herein, A549 cells were used as the experimental model in the present study as a means to intuitively mimic the potential effects of PM₁₀ on lung health. Furthermore, they have been used by the European Commission to address and explore PM components and their subsequent effects. In addition, the release of LDH was analyzed as a means to evaluate the effect of PM on cell integrity and viability, since the loss of intracellular LDH and its release into the culture medium are indicators of cell membrane damage leading to irreversible cell death^[18]. Reliability, speed, and simple evaluation are some of the advantages of this test^[19], which has been used as an indicator of cadmium chloride cytotoxicity in liver (HepG2) cells^[20].

Despite the efforts to ensure the accuracy and sensitivity of the tests performed, this study still has certain limitations. First, the PM₁₀ formulation (mixed with DPBS) and the concentrations used may differ from the PM commonly present in the atmosphere. Moreover, there are differences between the PM₁₀ data retrieved from the European Commission website and the results obtained for actual PM in the atmosphere. The PM₁₀ developed in the laboratory is synthetic, and its shape is relatively full and round, whereas the edges and corners of the PM₁₀ shape under natural conditions are more likely to break the cell membrane and affect cell ability. To overcome these limitations, additional comparative experiments should be performed in the future.

PM is a complex mixture of elemental carbon, sulfate, nitrate, ammonium, metals, and several other organic compounds^[21]. The viability of A549 cells significantly increases (12.3%) after metal removal from PM, indicating the important contributions of metal components to PM toxicity^{[22, 23]}. Exposure to PM₁₀ particles increases the production of tumor necrosis factor (TNF)-α in human monocytes and promotes apoptosis in A549 cells^[24]. This indicates that exposure to air contaminants induces an inflammatory state, modulates the immune system, and increases the expression of molecules that favor respiratory infections, thereby affecting the respiratory system^[13]. Therefore, based on these results, we will study the effects of heavy metals on the cellular activity, the release of LDH, and the inflammatory factors IL-6, IL-8, and TNF in human non-epithelial cells in the future. We will also further determine the relationship between air pollution and viruses responsible for major lung infections in recent years.

In this study, PM₁₀ at a low concentration (10 ng/L), close to the haze environment limit (150 μ g/m³ ), was considered. It was found that PM₁₀ affected the proliferation ability of A549 cells in a time-dependent manner at this low concentration. Therefore, the toxicity of PM₁₀ in A549 cells should be studied at various concentrations and times. The effect of low PM₁₀ concentrations on the incidence of lung cancer requires further investigation. This study provides additional evidence that low PM concentrations can considerably affect the viability of alveolar epithelial cells, thereby improving our understanding of the potentially negative effects of PM on the overall respiratory health of humans worldwide.

Ethics approval and consent to participate This study was approved by the Institutional Review Board Committee of the Dankook University (No. 2019-12-007) and was conducted in accordance with the Declaration of Helsinki.

Competing interests The authors declare no competing interests.

编辑：黄艳