Preliminary Evidence for a Hormetic Effect on DNA Nucleotide Excision Repair in Veterans with Gulf War Illness

Abstract

Introduction: Veterans of the 1991 Gulf War were potentially exposed to a mixture of stress, chemicals and radiation that may have contributed to the persistent symptoms of Gulf War Illness (GWI). The genotoxic effects of some of these exposures are mediated by the DNA nucleotide excision repair (NER) pathway. We hypothesized that individuals with relatively low DNA repair capacity would suffer greater damage from cumulative genotoxic exposures, some of which would persist, causing ongoing problems.

Materials and methods: Blood samples were obtained from symptomatic Gulf War veterans and age-matched controls. The unscheduled DNA synthesis assay, a functional measurement of NER capacity, was performed on cultured lymphocytes, and lymphocyte mRNA was extracted and analyzed by sequencing.

Results: Despite our hypothesis that GWI would be associated with DNA repair deficiency, NER capacity in lymphocytes from affected GWI veterans actually exhibited a significantly elevated level of DNA repair (p = 0.016). Both total gene expression and NER gene expression successfully differentiated individuals with GWI from unaffected controls. The observed functional increase in DNA repair capacity was accompanied by an overexpression of genes in the NER pathway, as determined by RNA sequencing analysis.

Conclusion: We suggest that the observed elevations in DNA repair capacity and NER gene expression are indicative of a "hormetic," i.e., induced or adaptive protective response to battlefield exposures. Normally such effects are short-term, but in these individuals this response has resulted in a long-term metabolic shift that may also be responsible for the persistent symptoms of GWI.

Keywords: DNA repair; Gulf War Illness; NER; exposure response; gene expression.

FIGURE 1

NER capacity as measured by the UDS assay of four veterans with Gulf War Illness (GWI) compared to nine age-matched controls, including one asymptomatic serviceman. NER capacities are expressed as a proportion of concurrently analyzed FF. Other controls are healthy, age-matched civilians and one asymptomatic veteran, as described in the Methods. As indicated by the asterisk, the affected population has a significantly increased mean DNA repair capacity (p = 0.016).

FIGURE 2

Unsupervised (left) and supervised (right) hierarchical clustering analysis of RNA sequencing data derived from lymphocytes of six veterans with GWI (Affected) and four controls, including one asymptomatic serviceman (Control). Both types of analysis distinguish the samples into two clusters, one cluster consisting of the symptomatic individuals and one cluster made up of the controls (the asymptomatic veteran is represented by the column second from left on both figures). The supervised analysis utilized gene expression data exclusively derived from the 20 canonical NER genes whose products are necessary to reconstitute activity in vitro.

FIGURE 3

RNA sequencing analysis of affected GWI veterans (gray bars) vs. age-matched controls (black bars) for each of the 20 canonical genes of the NER pathway. All gene expression levels are normalized to the mean of the controls. Overall, 12 of the 20 genes were relatively overexpressed in the affected GWI veterans. The elevations in expression of five of the genes are individually statistically significant: ERCC3 (p = 0.009), CCNH (p = 0.02), RAD23B (p = 0.005), RPA1 (p = 0.04), CDK7 (p = 0.0006).