CMA.

<p>Cellular Metabolic Activity assessed with the MTT-test after a 24 h exposure of three different cell culture setups (A549 and THP-1 monocultures and A549/THP-1 co-culture) to four doses (25, 75, 150 and 200 μg/ml) of particulate samples from the combustion of three different wood logs and wood pellets. Each bar represents the average of eight experiments. Whiskers indicate the standard error of the mean (SEM), asterisks indicate significance from unexposed control cells. <b>a</b> indicates significance from the birch log PM₁ sample, b indicates significance from the beech log PM₁ sample, <b>c</b> indicates significance from the spruce log PM₁ sample, <b>d</b> indicates significance from the pellet combustion PM₁ sample. <b>§</b> indicates significance from the A549 monoculture, <b>#</b> indicates significance from the THP-1 monoculture, <b>$</b> indicates significance from the A549/THP-1 co-culture.</p

Genotoxicity.

<p>DNA fragmentation in THP-1 cells after a 24 h exposure to four doses (25, 75, 150 and 200 μg/ml) of PM₁ samples from the combustion of three different wood logs and wood pellets expressed as percentage of DNA in the tail. Each bar represents the average of four independent experiments with 100 analyzed cells/assay + SEM of the experimental averages. Asterisks indicate statistical significance from blank control, <b>a</b> indicates significance from the birch log PM₁ sample, <b>b</b> indicates significance from the beech log PM₁ sample, <b>c</b> indicates significance from the spruce log PM₁ sample, <b>d</b> indicates significance from the pellet combustion PM₁ sample.</p

Oxidative stress.

<p>Oxidative stress assessed with the DCF-assay after a 24 h exposure of three different cell culture setups (A549 and THP-1 monocultures and A549/THP-1 co-culture) to four doses (25, 75, 150 and 200 μg/ml) of particulate samples from the combustion of three different wood logs and wood pellets. Each bar represents the average of eight experiments. Whiskers indicate the standard error of the mean (SEM), asterisks indicate significance from blank control. <b>a</b> indicates significance from the birch log PM₁ sample, <b>b</b> indicates significance from the beech log PM₁ sample, <b>c</b> indicates significance from the spruce log PM₁ sample, <b>d</b> indicates significance from the pellet combustion PM₁ sample. <b>§</b> indicates significance from the A549 monoculture, <b>#</b> indicates significance from the THP-1 monoculture, <b>$</b> indicates significance from the A549/THP-1 co-culture.</p

Cell membrane integrity.

<p>Cell membrane integrity assessed with the PI exclusion assay after a 24 h exposure of three different cell culture setups (A549 and THP-1 monocultures and A549/THP-1 co-culture) to four doses (25, 75, 150 and 200 μg/ml) of particulate samples from the combustion of three different wood logs and wood pellets. Each bar represents the average of eight experiments. Whiskers indicate the standard error of the mean (SEM), asterisks indicate significance from unexposed control cells. <b>a</b> indicates significance from the birch log PM₁ sample, <b>b</b> indicates significance from the beech log PM₁ sample, <b>c</b> indicates significance from the spruce log PM₁ sample, <b>d</b> indicates significance from the pellet combustion PM₁ sample. <b>§</b> indicates significance from the A549 monoculture, <b>#</b> indicates significance from the THP-1 monoculture, <b>$</b> indicates significance from the A549/THP-1 co-culture.</p

Spearman correlation of the chemical constituents with the toxicity endpoints.

<p>Spearman correlation of the chemical constituents with the toxicity endpoints.</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

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