In vitro exposures in diesel exhaust atmospheres: resuspension of PM from filters versus direct deposition of PM from air

Abstract

One of the most widely used in vitro particulate matter (PM) exposures methods is the collection of PM on filters, followed by resuspension in a liquid medium, with subsequent addition onto a cell culture. To avoid disruption of equilibria between gases and PM, we have developed a direct in vitro sampling and exposure method (DSEM) capable of PM-only exposures. We hypothesize that the separation of phases and post-treatment of filter-collected PM significantly modifies the toxicity of the PM compared to direct deposition, resulting in a distorted view of the potential PM health effects. Controlled test environments were created in a chamber that combined diesel exhaust with an urban-like mixture. The complex mixture was analyzed using both the DSEM and concurrently collected filter samples. The DSEM showed that PM from test atmospheres produced significant inflammatory response, while the resuspension exposures at the same exposure concentration did not. Increasing the concentration of resuspended PM sixteen times was required to yield measurable IL-8 expression. Chemical analysis of the resuspended PM indicated a total absence of carbonyl compounds compared to the test atmosphere during the direct-exposures. Therefore, collection and resuspension of PM into liquid modifies its toxicity and likely leads to underestimating toxicity.

Figure 1

Schematic diagram of the outdoor smog chamber, the laboratory sampling systems, and the biological exposure system (EAVES). Filters were collected directly under the chamber floor and processed post-collection for the exposures to resuspended PM. The diesel exhaust sample injection lines into the chamber are also depicted.

Figure 2

Gas chromatogram of VOC species in outdoor chamber. Top: after injection of exhaust from 1980 Mercedes (50 seconds) and 2.0 ppmC injection of SynUrb54 mixture, but before sunrise. Bottom: in dark after daylong sunlit reaction. The peak numbers correspond to the species listed in Table S1. Major compounds present in the chromatogram labeled injected were: 1 – iso-pentane, 10 – benzene, 19 – toluene, 23 – m-xylene, and 31 – 1,2,4-tri-methyl-benzene. After photochemical aging the species either completely reacted away or had significantly decreased.

Figure 3

Selective-ion chromatogram for the injections from the 1980 Mercedes and 2.0 ppmC of SynUrb54 in the chamber. of carbonyls from mister samples. Samples were collected after daylong irradiation. The peak numbers correspond to the species identified in Table S2. UR – Unreacted PFBHA solution, IS – Internal Standard, and some of the major peaks are 1 – formaldehyde, 3 – acetaldehyde, 6 – methyl ethyl ketone, 8– 2-pentanone, 10 – 2-hexanone, 14 – glyoxal, and 15 – methylglyoxal.

Figure 4

Selective-ion chromatogram of carbonyls from a water blank, a F12K media blank, and a filter sample taken after day-long irradiation of the injections from the 1980 Mercedes and 2.0 ppmC of SynUrb54 in the chamber. The peak numbers over the peaks correspond to the identities in Table S2. UR – Unreacted PFBHA reagent, IS – Internal Standard.

Figure 5

COX-2 and IL-8 m-RNA expression induced by exposure to directly deposited PM from the aged exposure in the 1980 Mercedes with SynUrb54 experiment and induced by resuspension exposures. The symbols * indicates statistically different from non-exposed incubator control; # indicates statistically significant difference compared to resuspension exposures. The error bars represent the mean ± standard error from the mean.

Figure 6

COX-2 and IL-8 m-RNA expression induced by exposure to directly deposited PM from the aged exposure in the 2006 Volkswagen with SynUrb54 experiment and induced by resuspension exposures. The symbols * indicates statistically different from non-exposed incubator control; # indicates statistically significant difference compared to resuspension exposures. The error bars represent the mean ± standard error from the mean.

Figure 7

COX-2 and IL-8 m-RNA expression induced PM from the AGED Exposure in the 1980 Mercedes with SynUrb54 experiment from resuspension exposures at concentrations of 2.65μg/cm² and 42.48μg/cm². The symbols * indicates statistically different from non-exposed incubator control; # indicates statistically significant difference compared to resus-pension exposures. The error bars represent the mean ± standard error from the mean.