Metabolic Profiling as Well as Stable Isotope Assisted Metabolic and Proteomic Analysis of RAW 264.7 Macrophages Exposed to Ship Engine Aerosol Emissions: Different Effects of Heavy Fuel Oil and Refined Diesel Fuel

Abstract

Exposure to air pollution resulting from fossil fuel combustion has been linked to multiple short-term and long term health effects. In a previous study, exposure of lung epithelial cells to engine exhaust from heavy fuel oil (HFO) and diesel fuel (DF), two of the main fuels used in marine engines, led to an increased regulation of several pathways associated with adverse cellular effects, including pro-inflammatory pathways. In addition, DF exhaust exposure was shown to have a wider response on multiple cellular regulatory levels compared to HFO emissions, suggesting a potentially higher toxicity of DF emissions over HFO. In order to further understand these effects, as well as to validate these findings in another cell line, we investigated macrophages under the same conditions as a more inflammation-relevant model. An air-liquid interface aerosol exposure system was used to provide a more biologically relevant exposure system compared to submerged experiments, with cells exposed to either the complete aerosol (particle and gas phase), or the gas phase only (with particles filtered out). Data from cytotoxicity assays were integrated with metabolomics and proteomics analyses, including stable isotope-assisted metabolomics, in order to uncover pathways affected by combustion aerosol exposure in macrophages. Through this approach, we determined differing phenotypic effects associated with the different components of aerosol. The particle phase of diluted combustion aerosols was found to induce increased cell death in macrophages, while the gas phase was found more to affect the metabolic profile. In particular, a higher cytotoxicity of DF aerosol emission was observed in relation to the HFO aerosol. Furthermore, macrophage exposure to the gas phase of HFO leads to an induction of a pro-inflammatory metabolic and proteomic phenotype. These results validate the effects found in lung epithelial cells, confirming the role of inflammation and cellular stress in the response to combustion aerosols.

Influence of wood species on toxicity of log-wood stove combustion aerosols: a parallel animal and air-liquid interface cell exposure study on spruce and pine smoke

“…For the Tox-ALI, the main flow of 5 L min − 1 was conditioned in a humidifier to approach 100% relative humidity while still staying below the condensation point at 37°C in a partial flow of 150 mL min − 1 , which led to the cell surfaces as a laminar flow. For OMICS-ALI systems, the diluted aerosol was conditioned to a relative humidity of 85% at + 37°C and directed through the exposure units at a flow rate of 100 mL min − 1 [39]. Following Tox-ALI exposure, A549 cells were incubated for 24 h, while OMICS-ALI endpoints were measured directly after the exposure.…”

“…The proteome was analysed from OMICS-ALI-exposed RAW264.7 cells using methods previously reported by Sapcariu et al [39]. A full description of the methods is given in the supplementary material, but briefly, the protein extracts of RAW264.7 cells were reduced and freed from sulfhydryl groups before the proteins were digested using sequencing grade endopeptidase LysC (Wako) and sequencing grade trypsin (Promega).…”

Comprehensive chemical comparison of fuel composition and aerosol particles emitted from a ship diesel engine by gas chromatography atmospheric pressure chemical ionisation ultra-high resolution mass spectrometry with improved data processing routines

“…8 In a similar experiment, cytotoxic effects were induced by the HFO or DF ship engine emissions in ALI-exposed murine macrophages. 9 Interestingly, the acute adverse biological effects of HFO and DF ship engine emissions were comparable, although the supposed toxicity of HFO emissions is much higher. Recent studies point out the importance of ship diesel particulate matter contribution, especially in coastal regions worldwide.…”

Emissions from a modern log wood masonry heater and wood pellet boiler: Composition and biological impact on air-liquid interface exposed human lung cancer cells

“…A new direction for studying direct exposures to emission aerosols has emerged with the appearance of air-liquid-interface (ALI) systems [24][25][26]. The ALI technology enables reproducible and direct on-site exposure of lung cell cultures to aerosols under realistic dilution, flow and humidity conditions [27] and can readily be combined with multi-omics analysis techniques [28,29].…”