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. 2019 Aug 12;8(5):731–740. doi: 10.1039/c9tx00112c

The chronic effect of amorphous silica nanoparticles and benzo[a]pyrene co-exposure at low dose in human bronchial epithelial BEAS-2B cells

Jing Wu a,†, Jie Zhang a,†, Jihua Nie a,†, Junchao Duan b, Yanfeng Shi b, Lin Feng b, Xiaozhe Yang b, Yan An a,✉, Zhiwei Sun b,✉
PMCID: PMC6762015  PMID: 31588350

graphic file with name c9tx00112c-ga.jpgAs the main components of fine particulate matter (PM2.5), silica nanoparticles (SiNPs) and benzo[a]pyrene (B[a]P) have attracted increasing attention recently.

Abstract

As the main components of fine particulate matter (PM2.5), silica nanoparticles (SiNPs) and benzo[a]pyrene (B[a]P) have attracted increasing attention recently. However, co-exposure to SiNPs and B[a]P causes pulmonary injury by aggravating toxicity via an unknown mechanism. This study aimed at investigating the toxicity caused due to long-term co-exposure to SiNPs and B[a]P on pulmonary systems at low dose using human bronchial epithelial (BEAS-2B) cells. The characterizations of SiNPs and B[a]P were done by transmission electron microscopy (TEM) and zeta potential granulometry. Cytotoxicity is evaluated using cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) activity; oxidative stress, cell cycle and apoptosis were assessed by flow cytometry, and inflammatory factors were detected using a Luminex xMAP system. Results show an obvious inhibition of cell proliferation and a marked increase in the LDH expression in the BEAS-2B cells after long-term co-exposure. Furthermore, long-term co-exposure is the most potent in generating intracellular ROS, thus causing inflammation. Cellular apoptotic rate is enhanced in the co-exposed group at low dose. Moreover, the long-term co-exposure induces significant cell cycle arrest, increasing the proportion of cells at the G2/M phase, while decreasing those at the G0/G1 phase. This study is the first attempt to reveal the severe synergistic and additive toxic effects induced by SiNPs and B[a]P co-exposure for long-term in BEAS-2B cells even at low dose.

Introduction

Fine particulate matter (PM2.5, the aerodynamic diameter of particles less than 2.5 μm) is considered as a major constituent of air pollutants and is brought into the atmospheric environment via multiple routes.1 The size and composition of PM2.5 play a decisive role in its detrimental effect. Craig et al. pointed out that PM2.5 is harmful to humans mainly because of their absorption of various environmental pollutants, such as organic compounds.2 However, the potential interactions of various components of PM2.5 responsible for these effects on health are still unclear.

Amorphous silica nanoparticles (SiNPs) are one of the most abundant inorganic elements in PM2.5 and are easier to be brought into the atmosphere via increasing applications in daily life.3,4 Yang et al. observed that the intratracheal instillation of SiNPs activates macrophages in the lung and leads to inflammation in BALB/c mice.5 Also, the in vitro studies indicate that SiNPs induce mitochondrial toxicity and cell dysfunction.6,7 SiNPs are observed in the lysosomes of target organs including lung, heart and liver.5 When inhaled or injected, SiNPs have adverse effects on human health owing to their small diameter, high dissolution rate, large surface area, etc.8 The lung is the primary target organ for inhaled particulate matters, such as SiNPs.9 Epidemiologic studies report that the highest lung cancer mortality among nonsmoking women in Xuanwei County is attributed to the higher concentration of indoor environmental pollutants, such as SiNPs and polycyclic aromatic hydrocarbons (PAHs).10 As confirmed by the International Agency for Research on Cancer (IARC), benzo[a]pyrene (B[a]P), a surrogate compound for the PAHs, is a human group 1 carcinogen being released into the ambient air due to incomplete fossil fuel combustion.11 Organic component analyses indicate that B[a]P is one of the major components in the organic pollutants in the PM2.5.12 Moreover, we have demonstrated that co-exposure to SiNPs and B[a]P for 24 hours can aggravate cytotoxicity and genotoxicity in BEAS-2B cells.13 Unfortunately, short-term assays cannot provide enough evidences to draw definite conclusions about the pulmonary damage caused by the SiNPs and B[a]P co-exposure. Using long-term combined exposures, we can determine cell dedifferentiation processes, which give an extra advantage over the short-term assays.14 Nevertheless, there are limited studies on long-term effects of pulmonary exposure to SiNPs when combined with B[a]P at low dose.

Hence, to gain a better understanding of the role of PM2.5 in pulmonary damage, BEAS-2B was chosen as a cell model to evaluate the toxic effects of SiNPs and B[a]P following a 30 passages long chronic co-exposure. In this study, we focused on the changes in inflammation, apoptosis and a series of toxic effects after individual or combined exposure to SiNPs and/or added B[a]P for long-term.

Materials and methods

Characterization of SiNPs and B[a]P

According to the Stöber method mentioned in a previous study, amorphous SiNPs were prepared in this experiment.7 First, ammonia, tetraethylorthosilicate (TEOS), ethanol and deionized water were mixed and stirred constantly for 12 h at 40 °C, 140 ± 10 rpm. Then, the mixture was collected and centrifuged for 15 min at 4 °C, 12 × 103 rpm. After centrifugation, deionized water was used to wash the SiNPs thrice. The particle size and distribution of SiNPs were measured using a transmission electron microscope (TEM) (JEOL, Japan) and the results were analyzed using the ImageJ software (National Institutes of Health, Bethesda, USA). Furthermore, based on dynamic light scattering (DLS), hydrodynamic size and zeta potential of SiNPs in different media were determined using a zeta electric potential granulometer (Malvern Instruments, Britain). Moreover, absorption, stability, purity and endotoxicity of the SiNPs and B[a]P co-exposure system were detected in our previous study.15 SiNPs were ultrasonicated for 5 min before the measurement. DMSO (≥99.7%, Sigma, USA) was used to dissolve B[a]P (>99.9%, Sigma, USA) solution in a required volume and the stock solution was stored in a refrigerator at 4 °C.

Cell culture and treatment

The human lung bronchial epithelial BEAS-2B cell line was donated by the Capital Medical University. The cells were cultured in a complete cell culture medium, which contained Dulbecco's modified Eagle's medium (DMEM; Corning, USA), 10% (v/v) fetal bovine serum (FBS; Gibco, USA) and 1% (v/v) penicillin/streptomycin, and was maintained in an incubator with 5% CO2 at 37 °C. To thoroughly investigate the combined toxic effects of SiNPs and B[a]P on the pulmonary system, the BEAS-2B cells were treated continuously with SiNPs or/and B[a]P for 30 passages. Moreover, to ensure the dispersion of nanoparticles in the culture medium, the SiNPs suspension was sonicated for 5 min each time before exposure.

Cytotoxicity

To assess the combined effect of SiNPs and B[a]P on cell viability, the cell counting kit-8 (CCK-8; Dojindo, Japan) assay was used in BEAS-2B cells. The cells were seeded in 96-well plates with 7 × 103 per well and exposed to different concentrations of SiNPs (2.5, 5, 10, and 20 μg mL–1) and B[a]P (2.5, 5, 10, and 20 μM) for 24 h. Then, the supernatants were removed and the cells were washed twice with phosphate-buffered saline (PBS). Subsequently, 100 μL fresh DMEM medium and 10 μL of CCK-8 solution were added to each well, followed by 2 h incubation at 37 °C. The absorbance of each group was determined at 450 nm wavelength using a microplate reader (Themo Multiskan MK3, USA). The no-observed-adverse-effect level (NOAEL) of SiNPs and B[a]P obtained were consistent with our previous studies and were used in the following co-exposure studies.15

Cell proliferation and LDH activity detection

After long-term co-exposure to SiNPs and B[a]P, the cell proliferation of BEAS-2B was examined using the CCK-8 (Dojindo, Japan) assay. The cell supernatants of each group were collected after long-term co-exposure for the lactate dehydrogenase (LDH) activity detection. A commercial assay kit of LDH activity (Jiancheng Bioeng Inst., China) was used, following the manual strictly. Samples were identified using a microplate reader (Themo Multiskan MK3, USA) at 450 nm absorbance wavelength.

Assessment of intracellular ROS

To evaluate the oxidative stress induced by SiNPs and B[a]P and to determine reactive oxygen species (ROS) generation in BEAS-2B cells, a fluorescent probe, 2′7′-dichlorofluorescin diacetate (DCFH-DA; Sigma, USA) was used as an intracellular oxidation-sensitive indicator. First, the BEAS-2B cells were seeded in 6-well plates after long-term co-exposure to SiNPs and B[a]P for 30 passages at low dose. Then, cell supernatants were removed and the cells were washed thrice using PBS. DCFH-DA working solution (10 μM) was dissolved in DMEM medium without serum, and co-incubated with the BEAS-2B cells for 30 min at 37 °C in dark. To eliminate the effects of unreacted DCFH-DA, cell media were removed and the cells were washed twice with DMEM without serum. Subsequently, BEAS-2B cells were harvested and centrifuged at 1200 rpm for 5 min at 4 °C. The supernatants were removed and the cells were resuspended in 0.5 mL PBS. The fluorescent intensities and percentages of positive cells were measured using a flow cytometer (Becton Dickinson, USA) at 488 nm excitation and 525 nm emission wavelengths.

Inflammation detection

Cytokines tumor necrosis factor-alpha (TNF-α) and monocyte chemotactic protein 1 (MCP-1) in BEAS-2B cells were measured using a commercial immunoassay kit (eBioscience, San Diego, USA) after long-term co-exposure for 30 passages. The operations were strictly performed according to the instructions of the manufacturer. The samples of each group were detected on a Luminex xMAP system (Luminex Corporation, Austin, USA) and analyzed using the ProcartaPlex software (eBioscience, USA).

Apoptosis analysis

Apoptosis in BEAS-2B cells was measured using a multispectral imaging flow cytometer. After co-exposure to SiNPs and B[a]P for 30 passages, BEAS-2B cells were washed twice with cold PBS and collected for centrifugation for 5 min at 2000 rpm. The supernatants were removed and 500 μL binding buffer was added to each sample for resuspending the cells. Then, the BEAS-2B cells were co-incubated with 5 μL fluorescein isothiocyanate-conjugated Annexin V antibody (Annexin V-FITC) and 5 μL propidium iodide (PI, KeyGen Biotech, China) at room temperature in dark for 15 min. Approximately 5000 cells were recorded using a high sensitivity multispectral imaging flow cytometer (Amnis, ImageStream, USA). The data were further analyzed with Flowjo 7.6 (Tree Star, USA) and IDEAS 6.0 (Amnis, USA).

Cell cycle analysis

Cell cycle in BEAS-2B cells was examined using a flow cytometer (Becton Dickinson, USA) after long-term co-exposure to SiNPs and B[a]P for 30 passages. Briefly, the BEAS-2B cells were harvested in a centrifuge tube and washed thrice with cold PBS, and then centrifuged at 1200 rpm for 5 min at 4 °C. The supernatant was removed and cold 70% ethanol was added to the cells. After incubation overnight at 4 °C, the BEAS-2B cells were centrifuged for 10 min, and then washed once with cold PBS. Subsequently, the supernatant was removed and RNase (100 μL per tube) was added into the cells. After incubation for 30 min at 37 °C, PI (400 μL per tube) was added into the cells, which still needed to be incubated in dark for 30 min at 4 °C. Fluorescence intensities in each group were measured using a flow cytometer (Becton Dickinson, USA).

Statistical analysis

All experiments were performed at least in triplicate and are expressed as a mean ± standard deviation (SD). Statistical differences among different groups were analyzed with the one-way analysis of variance (ANOVA), while p < 0.05 meant statistical significance. The factorial design and two-factorial ANOVA were used to evaluate combined interactions between SiNPs and B[a]P.16,17 Similar to the statistical description in the reports by Asweto et al. (2017)17 and Yu et al. (2015),18 the profile plots (interaction plots) was adopted to compare a marginal mean for exploring the interactions between-subjects and/or within-subject factors. Interaction plots were constructed by General Linear Model (GLM) command in SPSS to estimate marginal means. All the statistical analyses were performed using the SPSS 19.0 software (SPSS Inc., USA).

Results

Characterization of SiNPs

The morphology and average diameter of SiNPs were observed by TEM. As shown in Fig. 1A and B, amorphous SiNPs were almost spherical and uniformly dispersed. The particle size distribution was calculated using an Image J software with five hundred particles and the average diameter of individual SiNPs was 49 ± 6 nm. The DLS method was used to determine the hydrodynamic diameter (nm) and zeta potential (mV) in different exposure media of SiNPs (Table 1). The hydrodynamic diameter of SiNPs in ultrapure water was 74 ± 1 nm. However, the diameter of SiNPs increased after the addition of serum to DMEM. The particle hydrodynamic diameters were 86 ± 1 nm and 90 ± 2 nm in DMEM with 10% FBS (10% DMEM) and 10% DMEM with DMSO, respectively.

Fig. 1. Characterization of SiNPs. (A) Transmission electron microscopic image of SiNPs. (B) Size distribution of SiNPs (49 ± 6 nm). (C) Cell viability of SiNPs at different concentrations. (D) Cell viability of BEAS-2B cells exposed to SiNPs (5 μg mL–1), B[a]P (5 μM) and their mixture (5 μg/mL + 5 μM) for 24 h. Data are expressed as means ± S.D. from three independent experiments. *p < 0.05, compared with the control; #p < 0.05, compared with the individual exposure.

Fig. 1

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Table 1. The hydrodynamic diameter and zeta potential of SiNPs in different dispersion medium.

Medium Zeta potential (mV) Hydrodynamic sizes (nm)
0 h 6 h 12 h 24 h
Ultrapure water –76 ± 1 75 ± 1 73 ± 1 72 ± 1 74 ± 1
10% DMEM –32 ± 1 84 ± 1 82 ± 1 82 ± 1 86 ± 1
10% DMED + DMSO –28 ± 1 85 ± 1 86 ± 1 84 ± 1 90 ± 2
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Cytotoxicity

To evaluate the cytotoxic effects of the SiNPs and B[a]P co-exposure in BEAS-2B cells, cell viability was measured after exposure to SiNPs or B[a]P at different concentrations for 24 h. In our previous study, cell viability was detected in BEAS-2B cells after exposure to B[a]P (2.5, 5, 10, and 20 μM) for 24 h.13 To ensure the completeness of this study, we provide the following details. A moderate dose-dependent reduction on the cell viability of BEAS-2B cells was observed after exposure to SiNPs or B[a]P individually. In this study, 5 μg mL–1 of SiNPs and 5 μM of B[a]P were selected for the NOAEL dosage and toxic effects of long-term exposure assessment in BEAS-2B cells. Compared with the control, the cell viabilities of SiNPs (5 μg mL–1) and B[a]P (5 μM) in the BEAS-2B cells decreased to 94.97 ± 4.10 (Fig. 1C) and 92.62 ± 3.74%, respectively.13 The cell viability of the co-exposure group decreased to 86.67% (p < 0.05, Fig. 1D).

Cell proliferation and LDH activity

Cell proliferation capacity in the BEAS-2B cells was measured using CCK8 proliferation assay after long-term co-exposure to SiNPs and B[a]P. Compared with the control, a significant inhibition was determined on cell proliferation in the BEAS-2B cells after co-exposure (p < 0.05, Fig. 2A and B). In contrast, a synergistic increase in the LDH activity was observed in the co-exposure group compared to that in the control (Fig. 2C and D).

Fig. 2. Cell proliferation and LDH activity of BEAS-2B cells after SiNPs and B[a]P co-exposure for 30 passages. (A) Cell proliferation of BEAS-2B cells after SiNPs and B[a]P co-exposure for 30 passages. (B) Interaction plot showed an additive interaction between SiNPs and B[a]P on cell proliferation (F = 0.905, p = 0.360). (C) LDH activity of BEAS-2B cells after SiNPs and B[a]P co-exposure for 30 passages. (D) Interaction plot illustrated a synergistic interaction between SiNPs and B[a]P on LDH activity (F = 35.309, p = 0.000). Data are expressed as means ± S.D. from three independent experiments. *p < 0.05, **p < 0.01 compared with the control; ##p < 0.01, compared with the individual exposure.

Fig. 2

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Assessment of the intracellular ROS generation

To observe the change in oxidative stress induced by SiNPs and B[a]P co-exposure, ROS generation was measured using a flow cytometer. The results indicate that long-term co-exposure to SiNPs and B[a]P dramatically elevates ROS generation compared to that on individual exposure or the control (p < 0.05, Fig. 3A). Asynergistic interaction between SiNPs and B[a]P in BEAS-2B cells was obtained by a factorial analysis (Fig. 3B).

Fig. 3. Oxidative stress of BEAS-2B cells after SiNPs and B[a]P co-exposure for 30 passages. (A) Intracellular ROS generation of BEAS-2B after SiNPs and B[a]P co-exposure for 30 passages. (B) A synergistic interaction between SiNPs and B[a]P on ROS generation illustrated by interaction plot (F = 5.419, p = 0.048). Data are expressed as means ± S.D. from three independent experiments. **p < 0.01 compared with the control; #p < 0.05, compared with the individual exposure.

Fig. 3

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Inflammatory factor levels

To analyze changes in inflammation induced by long-term co-exposure, the levels of inflammatory factor TNF-α and MCP-1 were analyzed in BEAS-2B cells. As shown in Fig. 4, the data suggests that co-exposure to SiNPs and B[a]P results in a remarkable increase in both the TNF-α and MCP-1 levels compared to the control (p < 0.05). It is worth noting that TNF-α has an additive effect and MCP-1 has a synergistic effect in the co-exposure group, as obtained by the factorial analysis (Fig. 4B and D).

Fig. 4. Changes of inflammation in BEAS-2B cells after SiNPs and B[a]P co-exposure for 30 passages. (A) The expression of inflammatory factors TNF-α. (B) Interaction plot showed an additive interaction between SiNPs and B[a]P on the TNF-α expression (F = 1.042, p = 0.317). (C) The expression of MCP-1 (D) interaction plot illustrated a synergistic interaction between SiNPs and B[a]P on the MCP-1 expression (F = 29.708, p = 0.000). Data are expressed as means ± S.D. from three independent experiments. **p < 0.01 compared with the control; #p < 0.05, ##p < 0.01 compared with the individual exposure.

Fig. 4

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Detection of apoptosis

To observe apoptosis comprehensively, the apoptotic rate in BEAS-2B cells was measured using an imaging flow cytometer. The data indicated that BEAS-2B cells in the control group appear lower stained fluorescence ratio of Annexin V-FITC/PI and cells had integral shape (Fig. 5A). In contrast, co-exposure to SiNPs and B[a]P increases the stained fluorescence ratio and induces abnormal changes in the cell morphology of BEAS-2B cells (Fig. 5E). Compared with the control, the apoptotic rate in the BEAS-2B cells exposed to SiNPs or B[a]P show a significant increase (p < 0.05) (Fig. 5F). Moreover, the apoptotic rate in the co-exposure group was enhanced to 1.89-fold of the control (p < 0.05), which was much higher than that in the individual one. The factorial analysis showed an additive interaction between SiNPs and B[a]P in BEAS-2B cells after long-term co-exposure (Fig. 5G).

Fig. 5. Apoptosis of BEAS-2B cells after SiNPs and B[a]P co-exposure for 30 passages. (A) Control group, (B) DMSO group, (C) SiNPs-treated group, (D) B[a]P-treated group and (E) co-treatment group are the representative images of BEAS-2B cells apoptosis detected by an imaging flow cytometer. (F) The apoptotic rate of BEAS-2B cell measured by annexin V-FITC/PI assay. (G) Interaction plot illustrated an additive interaction between SiNPs and B[a]P on the cellular apoptotic rate (F = 1.521, p = 0.246). Data are expressed as means ± S.D. from three independent experiments. **p < 0.01 compared with the control.

Fig. 5

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Cell cycle arrest

Based on the statistical analysis, the proportion of BEAS-2B cells at the G0/G1 phase decreased markedly, while it elevated, significantly at the G2/M phase in the co-exposure group compared with that in the control (Fig. 6A and C). An additive effect was shown in BEAS-2B cells arrested at the G2/M phase after long-term co-exposure to SiNPs and B[a]P (Fig. 6B and D).

Fig. 6. G2/M phase cell cycle arrest of BEAS-2B cells after SiNPs and B[a]P co-exposure for 30 passages. (A) The percentage of cells in G0/G1 phase decreased and (C) the percentage of cells in G2/M phase increased significantly. (B) An additive interaction between SiNPs and B[a]P on G0/G1 phase (F = 0.180, p = 0.683)and (D) an additive interaction between SiNPs and B[a]P on G2/M phase were illustrated by interaction plots (F = 3.094, p = 0.117). Data are expressed as means ± S.D. from three independent experiments. *p < 0.05, **p < 0.01 compared with the control.

Fig. 6

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Discussion

Silica nanoparticles, the main component of PM2.5, have a high specific surface area and can absorb a large number of environmental organic pollutants, such as B[a]P.19 There are numerous research on the interactions among different components of PM2.5. Most of them focus on their acute toxic effects on health, without elucidating the complete exact mechanism.13,17,18 Therefore, we adopted a more practical approach to comprehensively interpret the potential mechanism of atmospheric PM2.5 on pulmonary damage. Herein, long-term co-exposure to low levels of SiNPs and B[a]P in BEAS-2B cells was chosen to investigate the toxic effects causing pulmonary damage. Our results have important significance in the future studies on the risk assessment of PM2.5.

Various specific characteristics of compounds can be responsible for their toxic effects in humans. Due to their smaller size and spherical shape, SiNPs have a large surface area for B[a]P absorption, leading to an increase in B[a]P bioavailability in cytoplasm.15 This property of SiNPs facilitates cellular uptake after co-exposure in BEAS-2B cells. Our previous study has indicated that the SiNPs and B[a]P co-exposure system has high absorption and thermal stability, as determined by Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA).15 In addition, the numerous hydroxyl radicals (˙OH) absorbed on the surface of SiNPs can cause more adverse effects in cells.18 Hence, we suggest that long-term co-exposure to SiNPs and B[a]P even at low dose induces more serious cellular dysfunction than exposure to the individual compounds, giving further evidence to its promotion of pulmonary damage.

Oxidative stress and inflammation are considered to be some of the adverse biological effects of nanoparticles both in in vivo and in vitro studies.20–22 It is worth noting that SiNPs can cause different biological effects on entering the cells. Among the possible effects induced by SiNPs, the intracellular generation of ROS has been reported by different authors. Nevertheless, most studies use high dose SiNP exposure to induce such type of effect. Positive ROS induction is observed in human hepatoma HepG2, after exposures to SiNPs at 25 μg mL–1.7 In human umbilical vein endothelial cells (HUVECs), an increased generation of ROS is observed after exposure to SiNPs at 25 μg mL–1.23 Moreover, we have previously shown that ROS generation was not a predominant response after short-term co-exposure to SiNPs and B[a]P at low levels.13,15 Nevertheless, it is interesting to note that long-term co-exposure to SiNPs (5 μg mL–1) and B[a]P (5 μM) synergistically elevates the ROS generation as demonstrated in our study even at low dose (Fig. 3). It indicates that the generation of ROS is not an inherent effect of SiNPs but is dependent on the exposure time and other compounds in the coexistent system. Furthermore, the excessive generation of ROS may inhibit cell proliferation via causing DNA damage or protein dysfunction.24 To confirm the BEAS-2B cell proliferation induced by the co-exposure, a CCK-8 assay was used in our study. Compared with SiNPs or B[a]P exposure alone, the co-exposed group has a significant inhibition on cell viability (Fig. 2A). Moreover, we also found a remarkable increase in the LDH activity in BEAS-2B cells after long-term co-exposure (Fig. 2B). These results indicate that long-term co-exposure to SiNPs and B[a]P inhibit cell proliferation and increase the cell membrane penetrability.

Disturbed redox homeostasis can induce the sustained stimulation of pro-inflammatory cytokines and chemokines, provoking or exacerbating chronic inflammation in humans.1 Our study indicates that the important pro-inflammatory cytokine TNF-α and chemokine MCP-1 were significantly increased in the co-exposure group than those in the control and the individual (Fig. 4). Consistent with our results, Duan et al. have reported that SiNPs and B[a]P co-exposure increases toxic effects by causing inflammatory response and blood hypercoagulable state in zebrafish embryos.15

Cell cycle is intimately connected with cell replication, division and proliferation, and can be affected by some adverse conditions, including DNA damage, abnormal DNA replication and nutrient depletion.25 Cell cycle distribution is usually used to determine and repair DNA damage, thus maintaining the genomic stability of cells.26 In order to provide extra time for repairing DNA damage, the cells are prevented from activating next stage mitosis at the G2/M phase.19 At present, our data demonstrates that cell cycle is arrested at the G2/M phase in BEAS-2B cells after long-term co-exposure to SiNPs and B[a]P. Cell cycle is one of the important elements to affect cell proliferation. Similar to our results, Asweto et al. have demonstrated that co-exposure to SiNPs and B[a]P can induce the G2/M phase arrest in HUVECs cells, which in turn can inhibit cell proliferation.17

Apoptosis is a classical mode of programmed cell death (PCD) that results in eliminating the damaged cells or cell organelles to maintain homeostasis in organisms. The abnormal regulation of apoptosis usually contributes to the occurrence of tumors.27 In this study, an imaging flow cytometer was used to detect the apoptotic rate that provided higher fluorescence sensitivity and better cell resolution than the ordinary one. Our results suggest that the excessive generation of ROS and inflammation induced by the long-term co-exposure to SiNPs and B[a]P led to redox imbalance associated with apoptosis. Moreover, Donaldson et al. have pointed out that the larger surface area and higher absorption of nanoparticles can enhance the health risks in humans.28 This special property of nanoparticles explains that co-exposure to SiNPs and B[a]P induces a higher apoptotic rate than the individual exposure in BEAS-2B cells (Fig. 5). Furthermore, co-exposure to SiNPs and B[a]P up-regulates Bax, Caspase-3 and Caspase-9 expressions while down-regulates Bcl-2 in the cells.17 The pro-apoptotic protein Bax enters into the cellular mitochondrial membrane to activate the p53-mediated apoptosis, which may be responsible for cancer.29,30 In addition, the imbalance of redox state and aggravation of DNA damage can affect cellular apoptosis.13,17 Our previous study has indicated that co-exposure to SiNPs and B[a]P for 24 h can induce oxidative stress and pulmonary damage, and can also increase multinucleation as well as DNA damage in BEAS-2B cells.13 Notably, DNA damage can activate the G2/M check point via a rapid response system.31 Hence, we assume that long-term co-exposure to SiNPs and B[a]P promotes apoptosis in BEAS-2B cells via the G2/M phase arrest, resulting in the inhibition of cell proliferation.

Conclusions

In summary, our results demonstrate that long-term SiNPs and B[a]P co-exposure at low dose inhibits cell proliferation and aggravates imbalance of oxidative state, inflammatory response, apoptosis and cell cycle induction in the BEAS-2B cells. Therefore, our results suggest the effect of combined SiNPs and B[a]P, present in PM2.5 air pollutants, on public health. To explore the potential mechanisms of the combination of SiNPs and B[a]P, further study needs to focus on molecular interactions and signaling pathways in in vitro and in vivo.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgments

This study was supported by the National Nature Science Foundation of China (81803271), China Postdoctoral Science Foundation (2018M642318, 2019T120458) and Jiangsu Planned Projects for Postdoctoral Research Fund (2018K234C).

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