Acute and Chronic Molecular Signatures and Associated Symptoms of Blast Exposure in Military Breachers

Abstract

Injuries from exposure to explosions rose dramatically during the Iraq and Afghanistan wars, which motivated investigations of blast-related neurotrauma and operational breaching. In this study, military "breachers" were exposed to controlled, low-level blast during a 10-day explosive breaching course. Using an omics approach, we assessed epigenetic, transcriptional, and inflammatory profile changes in blood from operational breaching trainees, with varying levels of lifetime blast exposure, along with daily self-reported symptoms (with tinnitus, headaches, and sleep disturbances as the most frequently reported). Although acute exposure to blast did not confer epigenetic changes, specifically in DNA methylation, differentially methylated regions (DMRs) with coordinated gene expression changes associated with lifetime cumulative blast exposures were identified. The accumulative effect of blast showed increased methylation of PAX8 antisense transcript with coordinated repression of gene expression, which has been associated with sleep disturbance. DNA methylation analyses conducted in conjunction with reported symptoms of tinnitus in the low versus high blast incidents groups identified DMRS in KCNE1 and CYP2E1 genes. KCNE1 and CYP2E1 showed the expected inverse correlation between DNA methylation and gene expression, which have been previously implicated in noise-related hearing loss. Although no significant transcriptional changes were observed in samples obtained at the onset of the training course relative to chronic cumulative blast, we identified a large number of transcriptional perturbations acutely pre- versus post-blast exposure. Acutely, 67 robustly differentially expressed genes (fold change ≥1.5), including UFC1 and YOD1 ubiquitin-related proteins, were identified. Inflammatory analyses of cytokines and chemokines revealed dysregulation of MCP-1, GCSF, HGF, MCSF, and RANTES acutely after blast exposure. These data show the importance of an omics approach, revealing that transcriptional and inflammatory biomarkers capture acute low-level blast overpressure exposure, whereas DNA methylation marks encapsulate chronic long-term symptoms.

Keywords: blast overpressure; epigenetics; sleep; tinnitus; traumatic brain injury.

FIG. 1.

(A) Protocol for operational breaching and blast exposure throughout the 10-day training course, showing pre- and post-blast exposure blood draws on days 1 and 10, respectively. (B) Distribution of number of lifetime operational blast exposures with history of self-reported TBI (red) and no history of TBI (blue). psi, pounds per square inch; TBI, traumatic brain injury. Color image is available online.

FIG. 2.

Differentially methylated regions (DMRs) associated with cumulative blast exposure. (A) DMRs with corresponding observed methylation data, showing gain (red) and loss (blue) of DNA methylation in groups with high relative to low lifetime exposure to blast events. (B) DNA methylation and gene expression changes, with the majority of genes showing the expected anticorrelation between DNA methylation and gene expression. (C) Genome browser representation of PAX8 antisense transcript expression relative to accumulative lifetime blast exposure in low (blue) versus high (yellow) exposed breacher groups. After an anticorrelated pattern of gain of DNA methylation and repression of gene expression, we see that the low accumulative blast-exposed group shows a loss of DNA methylation DMR track in blue, whereas the high accumulative blast-exposed group shows a gain of methylation indicated in red in the DMR track with coordinated repression of gene expression, as shown per subject. It should be noted that the DMR seems to be specifically regulating the PAX8 antisense transcript given that the upstream gene, PSD4, does not seem to be impacted by the differential methylation at the DMR. AS1, antisense RNA 1; DSCR3, Down syndrome critical region gene 3; FAM53A, family with sequence similarity 53 member A; LOC643387, TAR DNA-binding protein pseudogene; LogFC, log fold-change; NPHP1, nephrocystin 1; NRP2, neuropilin 2; PAX8, paired box gene 8; PSD4, pleckstrin and Sec7 domain-containing 4. Color image is available online.

FIG. 3.

Gene expression changes track molecular response to acute exposure to blast. (A) Volcano plot highlighting genes that passed multiple testing correction (6362 genes), that also showed greater than ±1.5 fold change in expression (336 genes, red). The y-axis shows the unadjusted p value. (B) Showing genes (67) with robust expression changes pre- versus post-blast training with fold change ≥|1.5| and excluding rarely expressed genes (logCPM <4). logCPM, log counts per million; LogFC, log fold-change. Color image is available online.

FIG. 4.

Symptom-associated DNA methylation analyses. (A) Differentially methylated genes associated with ringing in the ear/tinnitus in high relative to low blast-exposed groups, denoting genes implicated in auditory functioning by an asterisk (*). (B) Of note, KCNE1 and CYP2E1 genes follow the expected anticorrelated pattern of increase in DNA methylation with decrease in gene expression. CYP2E1, cytochrome P450 family 2 subfamily E member 1; DUSP12, dual specificity phosphatase 12; GPR148, G-protein-coupled receptor 148; KCNE1, potassium voltage-gated channel subfamily E member 1; LogFC, log fold-change; PGLYRP1, peptidoglycan recognition protein 1; PLXNB2, plexin B2; RGS14, regulator of G-protein signaling 14. Color image is available online.

FIG. 5.

Heatmap showing significant changes in cytokine level tracking with blast exposure (days 7 and 9) using day 4 (no exposure to blast activity) as the reference (Bonferroni corrected). EGF, epidermal growth factor; GCSF, granulocyte colony-stimulating factor; HGF, hepatocyte growth factor; MCP1, monocyte chemoattractant protein 1; MCSF, macrophage colony-stimulating factor; RANTES, regulated upon activation normal T-cell expressed and secreted. Color image is available online.