Additional file 1 of A novel in-situ method to determine the respiratory tract deposition of carbonaceous particles reveals dangers of public commuting in highly polluted megacity

Additional file 1. Experiment quality assurance and supplementary results. Table S1: List of related in situ respiratory tract deposition dose studies. Table S2: Summary of related in situ respiratory tract deposition studies using hydrophobic particles. Table S3: Mean DDR estimated using different assessment methods. Figure S1: Instrument laboratory intercomparison with reference system. Figure S2: Micro-aethalometer intercomparison in Leipzig, Germany. Figure S3: Micro-aethalometer intercomparison in Metro Manila, Philippines, using ambient street-site aerosol. Figure S4: Flow rate through dry and wet (after exposing to breath air) particulate filter. Figure S5: Descriptive statistics of measured parameters in TMEs between public transport and walking. Figure S6: Descriptive statistics of measured parameters separated between males and females. Figure S7: Deposition dose rate as a function of measured BC exposure concentrations

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

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

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

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