Objective To evaluate acute toxic effects of diesel engine exhaust (DEE) in human bronchial epithelial (16HBE) cells by an air liquid interface aerosol dynamic direct exposure method.
Methods The 16HBE cells seeded on porous membranes were exposed to DEE for 5, 10, 15, 20, 30 minutes at a flow rate of 20 mL/min, 37℃. Cell viabilities were evaluated by CCK-8 assay, lactic dehydrogenase (LDH) release assay, and neutral red uptake (NRU) assay, respectively. Apoptosis rates of cells with viability > 50% were determined by flow cytometry assay. Cells exposed to filtered clean air served as the control group and were applied the same exposure protocol as the treatment group.
Results The data of three assays determining cellular viability showed that cellular viabilities of the treatment groups after continuous exposure to DEE for 15, 20, 30 minutes were significantly reduced compared with the control group (P < 0.05), and possessed good time-response relationships. The damage of 16HBE cells after short-term exposure to DEE at a low flow rate was mainly cell membrane injury, followed by mitochondrial injury. Both the early apoptosis rates and the late apoptosis and necrosis rates of the treatment groups after exposure to DEE for 10, 15 minutes were significantly higher than those of the control group (P < 0.05). Of the apoptosis cells in the 10 and 15 min treatment groups, the late apoptosis and the necrosis cell counts were more than the early apoptosis cells (P < 0.05).
Conclusion Air liquid interface exposure method can be applied to in vitro toxicity studies of DEE and other harmful aerosols. The findings also indicate that DEE could lead to acute toxicity such as cell membrane damage, mitochondrial injury and apoptosis.
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