The Experimental Study Of Injury Of Coke Oven Emissions Organic Extracts Exposure On Bronchial Epithelial Cells

Objectives Coke oven emissions(coke oven emission, COE) is a major environmental pollutants released during coke production, which has a variety of mutagenic and carcinogenic polycyclic aromatic hydrocarbons. Chronic exposure to COE is prone to develop inflammation and cancer, and inflammation can promote the conversion to the growth and metastasis of tumor cells. In this study, we use different doses of organic extract of coke oven emissions exposure 24 h and 5d on 16 HBE cells. To explore the damage of bronchial epithelial cells treated with organic extract of coke oven emissions. In order to find candidate biomarkers of inflammatory injury induced by COE, to provide evidence and clues for the analysis of the COE exposed populations biomarker, to investigate the potential link between the inflammatory and immune damage and carcinogenic effects of coke oven emissions.Methods 1 Organic extract of coke oven emissions exposure 24 h on 16 HBE cells: the human bronchial epithelial cell line 16 HBE seeded in MEM medium containing 10% fetal bovine serum, subculture under the conditions of 37 ℃ and 5% CO2. When the cell fusion rate is 70% then treated with different concentrations of organic extract of coke oven emissions, the concentration is followed by 5mg/L, 10mg/L, 20mg/L, the fetal bovine serum concentration is 5%. Also set the COE vehicle control with 0.1% DMSO, per dose group is set up parallel samples.2 Organic extract of coke oven emissions exposure 5d on 16 HBE cells: the culture conditions and exposure dose same as 24 h models, continuous exposure 5d, every time replace new medium which containing COE, each dose group is set up parallel samples.3 To assess cytotoxicity by MTT and lactate dehydrogenase release assay test.4 To detect the reactive oxygen species release level using flow cytometry.5 To assess genetic damage of 16 HBE cells by comet assay.6 To detect the expression of IL-1β, IL-4, IL-6, IL-10, IL-17 A, IL-17 F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, γ-interferon(interferon-γ, IFN-γ), soluble cell surface CD40 ligand(s CD40L) and tumor necrosis factor-α(TNF-α) using by Human Th17 Cytokine Panel 15-Plex.7 To detect the expression levels of IκBα, P-IκBα, P65 and P-P65 protein using western blot method.Results 1 After 20 mg/L organic extract of coke oven emissions exposure 24 h on 16 HBE cells, the survival rate of 16 HBE cells was 89.38%(P<0.05), the relative LDH content was 147.68%(P<0.05). After 10 mg/L and 20 mg/L organic extract of coke oven emissions exposure 24 h on 16 HBE cells, the relative LDH content was 124.39% and 149.64%(P<0.01). It appears damage of 16 HBE cells treated with organic extract of coke oven emissions, and the toxicity increased with the exposure period increasing.2 The release of reactive oxygen species could be detected in both the 24 h and 5h models, the index value gradually increased with the increase of exposure dose(P<0.01). In the 24hmodel, the increased in multiples of mean cell fluorescence intensity compared with the control group in 5 mg/L dose group lower than the 5d model. An increase in 24 h model is 14.47 times multiple, in 5d model is 17.85 times multiple.3 16 HBE cells appear DNA damage in single cell gel electrophoresis test. After 20 mg/L organic extract of coke oven emissions exposure 24 h on 16 HBE cells, the percentage of tail DNA increased 83.51% compared with control group(P<0.01), olive tail moment index increased 76.47%(P<0.05). After 20 mg/L organic extract of coke oven emissions exposure 5d on 16 HBE cells, the percentage of tail DNA increased 89.28% compared with control group(P<0.01), olive tail moment index increased 117.07%(P<0.01).4 After organic extract of coke oven emissions exposure 24 h on 16 HBE cells, the expression of IL-10 in each group was 1.25±0.54、1.39±0.13 and(1.90±0.73) pg/m L, with a good dose-response relationship(r=0.98, P<0.05). The expression of IL-23 only in the 10 mg/L and 20 mg/L dose group. After organic extract of coke oven emissions exposure 5d on 16 HBE cells, the expression of IL-10 in each group was 1.71±0.02、1.49±0.13 and(2.82±0.16) pg/m L. The expression of IL-1β only in this model. It shows that organic extract of coke oven emissions can affect the expression of inflammatory factors.5 The expression of NF-κB pathway protein after organic extract of coke oven emissions exposure 24 h on 16 HBE cells: the expression of IκBα protein showed a downward trend, the amount of decrease in 5 mg/L, 10 mg/L and 20 mg/L dose groups were 26%, 33% and 56% compared with control group(P<0.05). The expression of P-IκBα protein in the 10mg/L and 20mg/L dose group increased significantly, the increase in 10mg/L group was 79% compared with the control group(P<0.05), the expression levels in 20 mg/L group was 2.02 times compared with control group the(P<0.05). The expression of P-P65 protein in 10 mg/L group was 3.84 times compared with control group the(P<0.05), in 20 mg/L group was 6.43 times compared with control group the(P<0.05). The expression of P65 appears no significant change.Conclusions 1 IL-23 and IL-1β are respectively reflect the 24 h and 5d inflammatory injury, IL-10 may be a good reaction of the inflammatory injury of coke oven emissions exposure 24 h and 5d on 16 HBE cells.2 Inflammatory and immune injury may be an early effect of carcinogenic changes of coke oven emissions.3 The damage of organic extract of coke oven emissions exposure on 16 HBE cells is reate with NF-κB signal activation.