[Toxicity of vehicle exhaust on BEAS-2B cells in vitro by air-liquid interface].

OBJECTIVE

To evaluate the toxic effect of vehicle exhaust( VE) on lung epithelial cells by air-liquid interface( ALI) method in vitro, and analyze the different toxicity of VE after being treated with 0. 2 μm filter.

METHODS

VE were collected using20 liter Tedlar bags and their particulate matter( PM) number, surface and mass concentration were measured by particle size spectrometer for the interference of 0. 2 μm filter or non-filter. Four groups were included, which divided into blank control group, clean air group, filtered VE exposure group, non-filtered VE exposure group. The blank control group did not do any treatment; the clean air group was an artificial gas containing21% O_2 and 79% N_2; the filtered VE group( marked as f VE) was filtered using a 0. 2μm particle filter for VE. The VE group was used VE directly collected by air bag and marked as non-f VE. Except the blank control group, BEAS-2B cells were treated with clean air or VE by ALI method at a flow rate of 25 mL/min, 37 ℃ for 60 min in vitro. Cell relative viability was evaluated by CCK-8 assay. The reactive oxygen species( ROS)generation was determined via flow cytometry with 2', 7'-dichloro-dihydro-fluorescein diacetate( DCFH-DA) probe. Apoptosis and necrosis rate were measured using the commercial kit of Annexin V-FITC/PI by flow cytometry.

RESULTS

In the non-f VE group, the PM of number, surface and mass concentration for 0. 5-10 μm diameters were 0. 24×103N/cm3, 0. 29 ×103μm2/cm3 and 0. 19 μg/m3, respectively, and for the PM of 10-500 nm diameters, they were 56 ×103N/cm3、34. 53 ×108nm2/cm3 and 95ng/m3, respectively. The PM of 0. 5-10 μm diameters in f VE group, their number, surface and mass concentration were less than 1 N/cm3, 1 μm2/cm3 0. 001 μg/m3, respectively. After filtration, the number, surface and mass concentration of PM in 10-500 nm diameters reduced by 89. 79%, 93. 57% and 90. 55%, respectively, as compared with non-f VE. In the clean air group, the cell relative viability, ROS generation, early apoptosis rate and late apoptosis and necrosis rate were( 90. 15 ± 4. 25) %, ( 1. 92 ± 0. 34)×105, ( 1. 09 ± 0. 48) % and( 8. 93 ± 3. 31) %, respectively. Compared with the clean air group, the cell relative viability, the ROS generation and the late apoptosis and necrosis rate of the two VE exposure groups were significantly different( all P < 0. 05). The cell relative viability of f VE exposure group were significantly higher than that in the non-f VE exposure group( t = 6. 331, P < 0. 001), and had no significant difference about the ROS generation[f VE ∶ non-VE =( 2. 94 ± 0. 21) ×105∶( 3. 32 ± 0. 49) ×10~5, t =-1. 252, P = 0. 279], early apoptosis rate [f VE∶ non-VE =( 1. 09 ± 0. 30) % ∶( 0. 99 ±0. 10) %, t = 0. 708, P = 0. 497] and late apoptosis and necrosis rate [f VE ∶ non-VE =( 21. 75 ± 10. 37) % ∶( 15. 32 ± 2. 74) %, t = 1. 347, P = 0. 242] between f VE and nonf VE exposure group( all P > 0. 05).

CONCLUSION

Increased toxicity of human lung cells( BEAS-2B) in vitro were observed by ALI method at a flow rate of 25 mL/min, 37 ℃ for60 min. After using a 0. 2 μm filter, the toxicity was obviously decreased.