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

The consumption of wood fuel is markedly increasing in developing and industrialized countries. Known side effects of wood smoke inhalation manifest in proinflammatory signaling, oxidative stress, DNA damage and hence increased cancer risk. In this study, the composition and acute biological impact of emissions of state-of-the-art wood combustion compliances: masonry heater (MH) and pellet boiler (PB) were investigated. Therefore A549 cells were exposed to emission aerosols in an automated air-liquid interface exposure station followed by cytotoxicity, transcriptome and proteome analyses. In parallel, aerosols were subjected to a chemical and physical haracterization. Compared to PB, the MH combustion at the same dilution ratio resulted in a 3-fold higher particle mass concentration (PM2.5) and deposited dose (PB: 27 $\pm$ 2 ng/cm2, MH; 73 $\pm$ 12 ng/cm2). Additionally, the MH aerosol displayed a substantially larger concentration of aldehydes, polycyclic aromatic hydrocarbons (PAH) or oxidized PAH. Gene ontology analysis of transcriptome of A549 cells exposed to MH emissions revealed the activation of proinflammatory response and key signaling cascades MAP kinase and JAK-STAT. Furthermore, CYP1A1, an essential enzyme in PAH metabolism, was induced. PB combustion aerosol activated the proinflammatory marker IL6 and different transport processes. The proteomics data uncovered induction of DNA damage-associated proteins in response to PB and DNA doublestrand break processing proteins in response to MH emissions. Taking together, the MH produces emissions with a higher particle dose and more toxic compounds while causing only mild biological responses. This finding points to a significant mitigating effect of antioxidative compounds in MH wood smoke

Particulate Matter from Both Heavy Fuel Oil and Diesel Fuel Shipping Emissions Show Strong Biological Effects on Human Lung Cells at Realistic and Comparable In Vitro Exposure Conditions

Background Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling. Objectives To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. Methods Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. Results The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon (“soot”). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. Conclusions Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices

Summary of the main HFO- and DF-particle exposure effects.

<p>The arrows indicate the direction of regulation for cellular functions derived from the most statistically significant enriched Gene Ontology terms from the transcriptome, proteome, and metabolome (details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126536#pone.0126536.s012" target="_blank">S2 Table</a>).</p><p>^x BEAS-2B up, A549 down</p><p>* BEAS-2B down, A549 up</p><p>Summary of the main HFO- and DF-particle exposure effects.</p

Experimental set-up and global omics analyses.

<p>(A) An 80 KW common-rail-ship diesel engine was operated with heavy fuel oil (HFO) or refined diesel fuel (DF). The exhaust aerosols were diluted and cooled with clean air. On-line real-time mass spectrometry, particle-sizing, sensor IR-spectrometry and other techniques were used to characterise the chemical composition and physical properties of the particles and gas phase. Filter sampling of the particulate matter (PM) was performed to further characterise the PM composition. Lung cells were synchronously exposed at the air-liquid-interface (ALI) to aerosol or particle-filtered aerosol as a reference. The cellular responses were characterised in triplicate at the transcriptome (BEAS-2B), proteome and metabolome (A549) levels with stable isotope labelling (SILAC and ¹³C₆-glucose). (B) Heatmap showing the global regulation of the transcriptome, proteome and metabolome.</p

Effects of shipping particles on lung cells.

<p>The net effects from the particles were referenced against the gaseous phase of the emissions. (A) Number of the regulated components in the transcriptome shows more genes regulated by the DF than the HFO particles (in BEAS-2B cells). Similar results were observed for the proteome (B) and metabolome (C) (in A549 cells). (D) Meta-analyses for the transcriptome and proteome using the combined Gene Ontology (GO) term analysis of the 10% most regulated transcripts and proteins. Individual GO terms are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126536#pone.0126536.s012" target="_blank">S2 Table</a>; the hierarchical pathways are indicated on the right. (E) Gene regulation of Wiki-pathway bioactivation; (F) gene regulation of Wiki-pathway inflammation; g, secreted metabolites; and h, metabolic flux measurements using ¹³C-labelled glucose. For all experiments, n = 3.</p

Chemical and physical aerosol characterisation.

<p>(A) The ship diesel engine was operated for 4 h in accordance with the IMO-test cycle. (B) Approximately 28 ng/cm² and 56 ng/cm² were delivered to the cells from DF and HFO, respectively, with different size distributions. The HFO predominantly contained particles <50 nm, and the DF predominantly contained particles >200 nm, both in mass and number. (C) Number of chemical species in the EA particles. (D) Transmission electron microscope (TEM) images and energy-dispersive X-ray (EDX) spectra of DF-EA and HFO-EA; heavy elements (black speckles, arrow); and contributions of the elements V, P, Fe and Ni in the HFO particles using EDX (* = grid-material). (E) Exemplary EA concentrations (right) and concentration ratios (left) for particulate matter-bound species. For all experiments, n = 3.</p