The toxicity of cooking oil fumes on human bronchial epithelial cells through ROS‐mediated MAPK, NF‐κB signaling pathways and NLRP3 inflammasome

Cooking oil fumes (COFs) are the main pollutants in kitchen and indoor air, which threaten human health. Exposure to COFs may lead to respiratory diseases and impair pulmonary function. To investigate the toxicity of COFs on human bronchial epithelial cells (Beas‐2B) and explore the underlying mechanisms, MTT assay was conducted to detect the viability of Beas‐2B. Intracellular reactive oxygen species (ROS) levels and nitric oxide (NO) levels were determined with DCFH‐DA assay and DAF‐FM assay. The expression of genes involved in inflammation were measured with quantitative real‐time PCR (qRT‐PCR). The phosphorylation and the expression of proteins related to Mitogen‐activated protein kinase (MAPK), NF‐κB signaling pathways were measured with western blot. Our results revealed that COFs decreased cell viability, increased the ROS levels and NO levels and induced apoptosis in Beas‐2B cells. The results of qRT‐PCR and western blot showed that the expression of NLRP3, p65, iNOS, IL‐1β, and the factors related to oxidative stress and inflammation increased, NF‐κB signaling pathway and MAPK signaling pathway were activated. This study provided some useful information to evaluate the toxicity of COFs and revealed the possible mechanism for the damage on respiratory system induced by COFs.

at higher risk for chronic bronchitis, for pregnant women, such exposure can also affect the fetus and increase the risks of adverse birth outcomes. [11][12][13] Studies have shown that exposure to COFs increased the risk of respiratory diseases, such as airway infection, chronic obstructive pulmonary disease, tuberculosis and asthma. [14][15][16][17] In addition, cardiovascular disease, abnormal pregnancy and cervical cancer are also related to COFs. 18,19 The occurrence of these diseases may be related to cytotoxic damage caused by COFs, it has been reported that the use of ventilation equipment can reduce the risk of cardiopulmonary death to 40% within 5 years. 20 It has been reported that the major compound contained in COFs can affect ROS and induce oxidative DNA damage in human lung carcinoma cells. 21 The apoptotic effect of COFs in fetal lung type II-like epithelium cells was investigated, which revealed that COFs induced apoptosis via mitochondrial intrinsic apoptosis pathway. 22 Trans, trans-2,4-decadienal (tt-DDE), a composition of cooking oil fumes, could induce oxidative stress and endoplasmic reticulum stress in human corneal epithelial cells. 23 But the toxicity of COFs on Beas-2B has rarely been reported.
The purpose of this study was to investigate the toxicity of COFs on Beas-2B cells, explore the signaling pathways in inflammation and oxidative stress and provide some experimental basis for studying the cytotoxic mechanism of COFs on respiratory system.

| Cooking oil fumes
COFs samples were collected by referring to Liu et al. 24 Briefly, The COFs were produced from heating peanut oil (200 ml) by an electric heater in iron pan and the temperature was maintained at the smoke point (280 ± 10 C). The fumes were collected with filter paper placed 50 cm upon the oil surface, which was connected to a vacuum pump.

| Cell signaling pathway analysis
Western blot analysis was carried out according to our previous study. 32 After treatment with COFs (0, 200, 400, and 600 μg/ml) for 24 h, Beas-2B cells were harvested and lysed, total protein extracts and nuclear extracts were prepared with total protein and nuclear protein extraction kits. Then the protein concentration was measured by BCA method. Protein samples were separated by 10% SDS-PAGE gels and transferred to PVDF membranes according to standard electroblotting procedures. After blocked with 5% non-fat milk for 1 h, the membrane was incubated with anti-Bax, anti-Bcl-2, anti-cleaved caspase-1, anti-cleaved caspase-3, anti-IκB, anti-p-IκB, anti-p65, anti-PCNA, anti-β-actin, anti-JNK, anti-ERK, anti-p38, antip-JNK, anti-p-ERK, anti-p-p38 monoclonal antibodies (at 1:2000 dilutions), then incubated with secondary antibodies. Finally, bands were observed by using an enhanced chemiluminescence kit. The relative level of total protein was normalized to β-actin and the relative level of nuclear protein was normalized to PCNA. 33,34

| Statistical analysis
The data were presented as means ± standard deviation (SD) at least three independently performed experiments. The data analyses were performed with one-way analysis of variance (ANOVA) with SPSS 21.0 (IBM SPSS, Armonk, NY, USA). p < .05 and p < .01 revealed statistical significance.

| PAHs in COFs
The PAHs in COFs were measured with GC/MS, the main content of PAHs in COFs was shown in Table 2.

| Oxidative stress induced by COFs in Beas-2B cells
The accumulation of ROS can reflect the degree of oxidative stress and play a key role in oxidative damage. 31,35 The effects of COFs on ROS production were detected with DCFH-DA fluorescence probe.
As shown in Figure 2, after the cells were treated with different concentrations of COFs (200, 400, and 600 μg/ml), with the increase of COFs concentration, the fluorescence intensity was 1.54, 2.04, and 2.51 times compared with the control group respectively. These results revealed that ROS levels in the cells increased in a dosedependent manner (p < .01).

| Inflammation induced by COFs in Beas-2B cells
The NO concentration can be regarded as an indicator of inflammatory reaction. 36 As shown in Figure 3, the fluorescence intensity in

| Apoptosis induced by COFs in Beas-2B cells
The apoptosis ratio was detected with flow-cytometry assay. 37 As shown in Figure 4, with the increase of COFs concentration (200, 400, and 600 μg/ml), apoptosis ratios were 10.85 ± 1.1%, 19.7 ± 1.3%, and 24.6 ± 1.8% respectively, which were significantly higher than that in the control group (5.6 ± 1.4%). Moreover, with the increasing concentrations of COFs, the percentages of apoptosis cells increased significantly and showed a dose dependent manner. These results indicated that Beas-2B cells proliferation was inhibited and apoptosis was activated after the treatment of COFs.

It is known that iNOS plays an important role in inflammation and
infection. 39 The result showed that the expression level of iNOS increased significantly (p < .01) after 200 μg/ml COFs treatment. In addition, the expression of NLRP3, IL-1β, IL-17, and other inflammatory factors were also increased (p < .01) ( Figure 5). 40,41

| Apoptosis-related proteins expression induced by COFs in Beas-2B cells
To further investigate the possible mechanism of apoptosis, the expression of Bcl-2-associated X (Bax), B-cell lymphoma-2 (Bcl-2), cleaved caspase-3, and cleaved caspase-1 were detected. 42 Compared with the control group, with the increase of COFs (200, 400, and 600 μg/ml), the ratio of Bax/Bcl-2 increased significantly (p < .01) ( Figure 6). When the concentration of COFs was 400 μg/ml and more, the levels of cleaved caspase-1 and cleaved caspase-3 proteins were increased compared with the control group significantly (p < .01). Furthermore, the level of cleaved caspase-3 in 600 μg/ml COFs-treated group was 2.51 times compared with the control group (p < .01).
These results showed that COFs increased the ratio of Bax/Bcl-2, the expression of cleaved caspase-3 and cleaved caspase-1 and induced apoptosis in Beas-2B cells.

| Effect of COFs on the MAPK signaling pathway
In order to further study the oxidative stress pathway mediated by COFs, the phosphorylation of extracellular signal-regulated kinase while p-p38/p38 significantly increased in the groups of 400 and 600 μg/ml compared with the control group respectively ( Figure 7B).

| Effect of COFs on the NF-κB signaling pathway
The expressions of p65 and IκB in COFs (200, 400, and 600 μg/ml) groups were measured to assess the contribution of the NF-κB pathway to the inflammation react in Beas-2B. 200, 400, and 600 μg/ml COFs upregulated the rate of p-IκB/IκB significantly (p < .01) ( Figure 8). Furthermore, it could be seen that with the increase of COFs concentration, the translocation of p65 in nucleus increased (p < .01) and the change of total p65 was not significant, indicating that NF-κB signaling pathway was activated.

| DISCUSSION
Lung is vulnerable and sensitive to exposure to air pollutants.
According to a Norwegian survey, employees in restaurant exposed to oil fumes were more likely to die from respiratory diseases such as asthma and emphysema. 15 Exposure to COFs increased the probability of acute respiratory infection in children according to a study in Thailand. 43  Beas-2B exposed to COFs was measured with q PCR. Data were expressed as means ± SD from three independent experiments. **p < .01 compared with the control group that exposure to COFs caused oxidative DNA damage and lipid peroxidation. 44 Lungs of mice after inhalation of COFs exhibited diffuse cellulose exudation from alveolar and bronchus, severe hemorrhage and moderate interstitial lymphoplasmacytic infiltration. 45 However, the cytotoxicity induced by COFs in Beas-2B cells has rarely been reported. In this study, the effect of COFs on oxidative stress, inflammation and apoptosis was explored, and the preliminary mechanism of COFs toxicity on Beas-2B cells was investigated.
Oxidative stress is a common manifestation of cell damage. 46 ROS produced by environmental factors can cause nuclear and apoptosis. [47][48][49] After COFs treatment, the accumulation of ROS in Beas-2B cells was significantly increased, which was similar to the damage of deoxynivalenol on HT-29 cells. 50 MAPK signaling pathway is closely related to oxidative stress and promotes the process of cell apoptosis, including ERK, JNK and p38 MAPK. 51 ROS accumulation can activate MAPK pathway and induce oxidative stress. 26 The phosphorylation of ERK, JNK and p38 was upregulated significantly after COFs treatment, suggesting that the MAPK signaling pathway in Beas-2B cells were activated, which was consistence with the mechanism about the toxicity of copper in mice. 52 Therefore, the mechanism of oxidative stress induced by COFs may be the activation of MAPK signaling pathways.
Inflammation is the primary factor to evaluate the effect of particles in the air on human health. 53  invasion or tissue damage. 59 Our results showed that the concentration of NO increased, inflammatory reaction occurred and inflammatory factor IL-1β, IL-6, IL-8, IL-17, IL-18, TNF-α, and CRP were upregulated after COFs treatment, and this result was consistent with the inflammation induced by PM 2.5. 60 NLRP3 is the most typical and widespread inflammation in pulmonary inflammatory diseases. 23 The activation of NLRP3 inflammatory complex is important to fight against respiratory tract infection, however, over activation may lead to serious diseases. 41 p65 is an important transcription factor regulating cell growth, proliferation, apoptosis and carcinogenesis. 25 It has been reported that cigarette smoke could increase the NLRP3 inflammatory complex and activate NF-κB pathway in THP-1 cells. 61 In this study, the distribution of p65 in the nucleus increased and the phosphorylation of IκB increased after COFs treatment, and this result was consistent with the toxicity induced by acrylamide in HepG2 cells. 62 The mechanism of COFs inducing inflammatory response of Beas-2B cells may be to activate NF-κB signaling pathway.
Apoptosis is programmed cell death and plays an important role in maintaining cell homeostasis. 51 Apoptosis is also regulated by many genes, the Bcl-2 is a potent inhibitor of apoptosis and plays a regulatory role in the process of cell apoptosis. 63 The proportion of proapoptotic members to the level of Bax in mitochondria is closely related to cell survival or apoptosis. 64 The ratio of Bcl-2 to Bax determines the cell's life and death, Bcl-2 family proteins transfer from the cytoplasm to the mitochondrial membrane and mediate the activation of caspase, mediating the mitochondrial endogenous apoptosis pathway. 26,65 It has been reported that crotonaldehyde could induce Beas-2B cells apoptosis by regulating the expression of caspase family proteins. 28 In this experiment, COFs could significantly upregulate the expression of Bax, cleaved caspase-1, cleaved caspase-3, decrease the expression of Bcl-2, the same mechanism was also addressed in the apoptosis caused by chlorogenic acid in chondrocyte cells. 66 COFs could induce apoptosis by regulating the mitochondrial apoptosis pathway.

| CONCLUSION
COFs exposure led to the decrease of cell viability, the accumulation of ROS and NO, oxidative stress, inflammation and apoptosis. The abnormal expression of apoptosis-related proteins Bax, Bcl-2, cleaved caspase-1, and cleaved caspase-3 in Beas-2B cells revealed that mitochondria-mediated intrinsic apoptosis pathway was activated.
COFs increased the phosphorylation of related proteins in MAPK family JNK, ERK, and p38 and led to oxidative stress ( Figure 9). COFs exposure increased the phosphorylation of IκB and promoted p65 translocation into nuclear and activated NF-κB signaling pathway ( Figure 9). This study gave a new understanding of the toxicity of COFs on Beas-2B cells and provided scientific data to assess its potential risks on lung disease induced by COFs.
F I G U R E 9 COFs induced Beas-2B cells damage through oxidative stress, inflammation and activated mitochondriamediated intrinsic apoptosis pathway ACKNOWLEDGMENTS