Proteomic, Metabolomic, and Lipidomic Analyses of Lung Tissue Exposed to Mustard Gas

Abstract

Sulfur mustard (HD) poses a serious threat due to its relatively simple production process. Exposure to HD in the short-term causes an inflammatory response, while long-term exposure results in DNA and RNA damage. Respiratory tract tissue models were exposed to relatively low concentrations of HD and collected at 3 and 24 h post exposure. Histology, cytokine ELISAs, and mass spectrometric-based analyses were performed. Histology and ELISA data confirmed previously seen lung damage and inflammatory markers from HD exposure. The multi-omic mass spectrometry data showed variation in proteins and metabolites associated with increased inflammation, as well as DNA and RNA damage. HD exposure causes DNA and RNA damage that results in variation of proteins and metabolites that are associated with transcription, translation and cellular energy.

Keywords: DNA and RNA damage; histology; inflammation; lipids; lung; mass spectrometry; metabolites; proteins; sulfur mustard.

Figure 1

Histology changes associated with HD exposure in EpiAirway^TM tissues. Paraffin-embedded sections from EpiAirway^TM tissues exposed to media or HD (0.1 mg/mL; 0.629 mM) were rehydrated and stained with hematoxylin and eosin.

Figure 2

Effects of HD on the viability of EpiAirway^TM tissues. Open bars represent mean ± SEM of cellular viability of untreated EpiAirway^TM tissues at 3 h (A) or 24 h (B).Closed bars represent mean ± SEM of cellular viability of EpiAirway^TM tissues at 3 h (A) or 24 h (B) following treatment with increasing concentrations of HD (0.01–2.5 mg/mL; 0.0629–15.7 mM). Cross-hatched bars represent mean ± SEM of cellular viability of EpiAirway^TM tissues at 3 h (A) or 24 h (B) with an equivalent amount of vehicle (corn oil). Checkered bars represent mean ± SEM of cellular viability of EpiAirway^TM tissues treated with 10% formalin (positive control) at 3 h (A) or 24 h (B). Experimental n is indicated within each bar. * Significantly decreased compared to vehicle control at p < 0.05. ** Significantly decreased compared to vehicle control at p < 0.01. SEM indicates the standard error of the mean.

Figure 3

Volcano Plots of proteins at (A) 3 h post exposure and (B) 24 h post exposure. Principal component analysis (PCA) plots for (C) exposed and control samples at 3 h post exposure and (D) exposed and control samples at 24 h post exposure.

Figure 4

Chromosomal mapping of down regulated proteins at the 3 h mark (A) and the 24 h mark (B). This shows a wide chromosomal effect of HD exposure over time leading to the dysregulation of multiple proteins.

Figure 5

Bar graphs of the grouped abundances of (A) plasminogen activator inhibitor 2 (PAI2) expression profile across the control groups and the 3 h and 24 h exposures, (B) Urokinase plasminogen activator surface receptor dysregulation at the 3 h and 24 h marks indicating an upregulation in recruitment of urokinase-type plasminogen activator (PLAU) at the 24 h timepoint and initial inflammatory response at associated with the decrease in uPAR. The dysregulation appears to increase post exposure to a point of significant dysregulation, (C) PLAU shows increase expression indicating an inflammatory response. Significant dysregulation (adj. p-value ≤ 0.05) is shown by **.

Figure 6

Bar graphs of the normalized abundances of (A) TIMP1 expression profile across the control groups, 3 h and 24 h exposures, (B) MMP-9 at the 3 h and 24 h marks, and (C) Ratios between the expression levels of MMP-9 vs. TIMP1. Significant dysregulation of TIMP1 and MMP9 (adj. p-value ≤ 0.05) is shown by * and significant changes in the ratio (p-value ≤ 0.05, students t-test) are indicated by *.

Figure 7

Pathway network mapping of enriched pathways upregulated (A) and downregulated (B) at the 24 h mark. Darker nodes are more significantly enriched gene sets. Bigger nodes represent larger gene sets. Thicker edges represent more overlapped genes.

Figure 8

Base Excision Repair (BER) Pathway expression profiles of the (A) 3 h exposure and the (B) 24 h post exposure.

Figure 9

mRNA Surveillance pathway expression mapping at the (A) 3 h and (B) 24 h post exposure levels.

Figure 10

Portion of nucleotide metabolism pathway around adenine metabolism—green is down-regulated and red is up-regulated.

Figure 11

Box-and-whisker plots of (A) sphingomyelin and (B) ceramide.