Cytotoxicity Assessment of PM2.5 Collected from Specific Anthropogenic Activities in Taiwan

Fine particulate matter (PM2.5) from different sources with different components have different health impact. In this research in Taiwan, composition and cytotoxicity of PM2.5 from long-range transport event (LRT), traffic activity, and outdoor cooking at night market were studied. The PM2.5 mass concentrations were 39.0 μg/m3 during LRT, 42.9 μg/m3 at traffic area, and 28.3 μg/m3 at the night market. Traffic area had highest concentrations of PCDD/Fs (46.9 fg I-TEQ/m3) when highest PAH concentrations of 3.57 BaPeq-ng/m3 were found at night market area. One quarter of PM2.5 mass at LRT and night market was constituted by water-soluble ion (26.02–28.93%). Road dust (represented by high concentration of Al and Ca) was the main contributor for metal element at traffic station whereas presence of natural salt (Na and Cl elements) was a marker of LRT and cooking activities. Cell viability reduced 9% after exposure to organic extracts of 0.316 μg of PM2.5 from LRT and night market samples. 150% elevation of ROS production was observed after exposure with organic compound of night market samples at the dose equivalent to 10.0 μg PM2.5. Organic extracts from night market induced positive genotoxicity in umu test (at a dose of 20.0 μg PM2.5).

A549 cell line was cultured in a 10 cm cell culture dish (dish), using Ham's F12 medium and adding 10% Fetal Bovine Serum (FBS) and 1% Penicillin / Streptomycin (P / S) An antibiotic is cultured in an environment of 37 ° C and 5% CO2 controlled humidity, and the cells are subcultured every 2 to 3 days and the medium is changed.
The cells must be washed twice with PBS to remove the serum components in the culture medium for trypsin action. After rinsing the cells with PBS, completely cover the cells attached to the bottom of the culture plate with 1 mL of 0.05% trypsin / EDTA (trypsin / EDTA). After 5 minutes of treatment, tap the culture plate to remove the cells from the bottom of the plate. Trypsin / EDTA was re-dissolved in a 1: 1 medium to stop the enzyme reaction, and then placed in a centrifuge at 12000 rpm for 3 minutes to remove the trypsin-containing medium. After resuspending in 10 mL of medium, take an appropriate amount. Excessive release of free radicals by cells under stimulation is a key factor in the generation of oxidative stress. In order to determine the amount of free radicals generated after the cells are exposed to fine suspended particulate extracts, that is, their potential oxidative stress, they will be cultured in complete medium (F12 medium containing 10 % FBS and 1% P / S) A549 cells were exposed to PM2.5 extract for 6 hours. After removing the exposure solution, the cells were exposed to 5 μM 2 ', 7'-dichlorodihydrofluorescein diacetate (DCFDA) superoxide. Anion determination of fluorescent dyes, cultured for 30 minutes in the dark, oxidized and metabolized DCFDA to DCF (2,7-dichlorofluorescin) with fluorescence, and the intensity of the fluorescence could reflect reactive oxygen species (ROS) When the content is high or low, the fluorescence intensity of cells exposed to PBS solution is measured by flow cytometry, and the oxidative stress index ROS level (BD, FACSVerse system, CA) is compared with the control group under the exposure of pollutants. , USA).
Test operation steps: 1. Expose the sample to 5 × 105 cells / 6 well-plate, while exposing the positive control and blank sample control 2. After 6 hours, remove the exposure solution and wash it once with PBS.
3. Expose at 0.05% Trypsin / EDTA for 5 minutes to transfer cells into a 15 mL centrifuge tube 4. After the cells are transferred into the centrifuge tube, rinse with PBS twice to remove the serum components present on the cells.
5. After exposure to DCFDA for 30 minutes in the dark, rinse twice with PBS 6. The fluorescence value (485nm-530nm) was measured by flow cytometry on the machine.

Umu test
Umu test was used for genotoxicity testing in this study (Shimada et al., 1994).
The genotoxicity of the fine suspension particulate extracts will be tested using the S.
typhimurium TA 1535 / pSK1002 and NM2009 strains in the presence of the recombinant cytochrome P450 CYP1A1 monooxygenase system.
Dissolve in different fine suspended particulate extracts using DMSO or a suitable solvent to make a solution, and if necessary, filter insoluble matter. The solution was added to the activated strain, and the medium was cultured at 37 ° C, and the strain was exposed to the fine suspension particulate extraction solution for 2 hours. The blank control group was prepared by simply exposing the solvent to the strain. The genotoxicity in the presence or absence of the recombinant cytochrome CYP 1A1 monooxygenase system was compared. If DMSO is used as the solvent to prepare the exposure solution, the final concentration of DMSO needs to be less than 1% (v / v).
In order to consider the possibility that the test object affects the growth of bacteria, the reaction bacterial solution is measured at an absorbance of 600 nm (A600 nm).
The β-galactosidase activity expressed by genotoxic activation was measured using 2-nitrophenyl-β-d-galactopyranoside as the target substance, and umu gene proliferation was used as the bacterial growth situation to standardize. As a result of the measurement, the expression of the β-galactosidase enzyme is determined by the coloration of the receptor when it is excited, and the absorbance (A420nm) is measured at a wavelength of 420nm after the end of the test step. When the test substance caused β-galactosidase expression activity to be more than 1.5 times higher than that of the blank control group, it was judged to be genotoxic.