The efficiency of EURO 6d car particulate filters is compromised by atmospheric aging: In vitro toxicity of gasoline car exhaust

Abstract

Tailpipe emissions from road traffic contribute substantially to the burden of fine inhalable particulate matter (PM_2.5) and deteriorate air quality. Exhaust emission standards, forcing improvements in combustion and exhaust after-treatment technology, considerably decreases combustion-related PM_2.5 emitted by modern cars. A549 cancerous alveolar and BEAS-2B normal bronchial epithelial cells were exposed at the air-liquid interface to the total aerosol or gas phase of either fresh or photochemically aged tailpipe emissions from a gasoline EURO 6d car equipped with a gasoline particulate filter. Diluted fresh emissions contained particle number concentrations comparable to low ambient air levels and induced no detectable cytotoxicity. Photochemical aging led to the formation of secondary aerosols and caused significant cytotoxicity. While the aged aerosol induced significant DNA damage, oxidative stress was more associated with volatile secondary species. Our results call for the consideration of the exhaust emission atmospheric transformation processes in future emission standards toward health effect-driven emission regulations.

Fig. 1.. PN concentrations and compositions in diluted tailpipe emissions.

(A) PN size distributions and (B) total PN concentrations derived from SMPS. Shaded area denotes the SD of four experiments (n = 4). (C) Refractory particle components; OM, nitrate (NO₃), ammonium (NH₄), and sulfate (SO₄) from AMS, and two quantities representing oxidative particle properties from OOPAAI (OP_AA) in equivalents of dehydroascorbic acid (DHA-eq.) (80) and OPROSI (ROS_DCFH) in equivalents of H₂O₂ (79). ROS_DCFH and OP_AA could not be quantified in fresh exhaust particles due to concentrations below the limits of quantification. Insert in (C): Oxygen-to-carbon (O:C) and hydrogen-to-carbon ratio (H:C) in OM. Because of OM concentrations close to the limit of quantification, elemental ratio in fresh exhaust particles could not be determined.

Fig. 2.. Compositions and concentrations (in ppb v/v) of gases and VOCs in diluted car exhaust.

(A) The concentration of carbon monoxide (CO), nitric oxide (NO), nitric dioxide (NO₂), ammonia (NH₃), methane (CH₄), and NMHCs in fresh exhaust derived by Fourier transform infrared spectroscopy; nitrogen oxides (NO + NO₂ = NOx) in the aged exhaust were measured by a chemiluminescence gas analyzer. (B) Concentrations of the five most abundant volatile ArHCs from PTR MS, REMPI MS, and GC-MS. Benzene and toluene could not be determined by offline sampling; hence, their concentrations from PTR measurements were added for total ArHC concentration (ΣArHC). (C) Representative PTR mass spectrum of the gas-phase composition. The sum formula refers to the composition of the VOCs in the exhaust, which are detected as protonated species. All quantities are corrected for a dilution of 17 used for the in vitro exposure experiments. PTR mass spectra of all experiments may be found in fig. S5.

Fig. 3.. Cytotoxic effects of fresh and aged gasoline car exhaust emissions on lung epithelial cells.

Alveolar A549 and bronchial BEAS-2B epithelial cells were exposed at the ALI to 1:17 diluted fresh or aged gasoline car exhaust emissions for 4 hours. Directly after exposure, metabolic activity (A) was measured by the reduction of resazurin into resorufin, and cytotoxicity (B) was measured by the release of LDH. The metabolic activity and cytotoxicity of exposed cells is compared to cells exposed to CA or to cells left in the IC as negative controls. Results are presented as means ± s.e.m. One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used for multiple comparisons. *P < 0.05, ***P < 0.001, and ****P < 0.0001. ns, not significant.

Fig. 4.. DNA damage in lung epithelial cells induced by gasoline car exhaust emissions.

Alveolar A549 (A to C) and bronchial BEAS-2B (D to F) epithelial cells were exposed at the ALI to total aerosol (TA) or gas phase (GP) of 1:17 diluted fresh or aged gasoline car exhaust emissions for 4 hours, and DNA damage was measured by Comet assay. DNA strand breaks were assessed by the alkaline version of the comet assay in A549 cells (A) and BEAS-2B cells (D). Positive controls (PC) = 50 μM H₂O₂. Incubator control, (IC). Oxidative DNA damage was detected in A549 (B and C) and BEAS-2B (E and F) cells with Fpg- and hOGG1-modified comet assay. Positive controls (PC) = 1.5 mM KBrO₃. Clean air, (CA). Bars represent means ± s.e.m. from three independent experiments. One-way ANOVA with Tukey’s post hoc test was used for multiple comparison. *P < 0.05 and **P < 0.01. Unpaired t test was used for comparison of two conditions (NC versus PC, fresh versus aged, and TA versus GP). Statistical significance is then indicated by #.

Fig. 5.. Proinflammatory and oxidative stress responses induced by gasoline car exhaust emissions in lung epithelial cells.

Alveolar A549 and bronchial BEAS-2B cells were exposed to the ALI to total aerosol (TA) or gas-phase (GP) of 1:17 diluted fresh or aged gasoline car exhaust for 4 hours. After exposure, basolateral medium from the exposed cells were collected for later measurements. IL-8 and IL-6 cytokine release was measured by enzyme-based immunosorbent assay (A to C). MDA release was measured by LC-MS/MS (D). Cells were collected, and the ratio of GSSG/GSH was measured by LC-MS and compared to negative controls, PC: 100 μM menadione (E). Bars represent means ± s.e.m. from three or four independent experiments. One-way ANOVA with Tukey’s post hoc test was used for multiple comparison. *P < 0.05 and ****P < 0.0001. Unpaired t test was used for comparison of two conditions (NC versus PC, fresh versus aged, and TA versus GP). Statistical significance is then indicated by #.