Impact of GADD45A on Radiation Biodosimetry Using Mouse Peripheral Blood

Abstract

High-dose-radiation exposure in a short period of time leads to radiation syndromes characterized by severe acute and delayed organ-specific injury accompanied by elevated organismal morbidity and mortality. Radiation biodosimetry based on gene expression analysis of peripheral blood is a valuable tool to detect exposure to radiation after a radiological/nuclear incident and obtain useful biological information that could predict tissue and organismal injury. However, confounding factors, including chronic inflammation, can potentially obscure the predictive power of the method. GADD45A (Growth arrest and DNA damage-inducible gene a) plays important roles in cell growth control, differentiation, DNA repair, and apoptosis. GADD45A-deficient mice develop an autoimmune disease, similar to human systemic lupus erythematosus, characterized by severe hematological disorders, kidney disease, and premature death. The goal of this study was to elucidate how pre-existing inflammation in mice, induced by GADD45A ablation, can affect radiation biodosimetry. We exposed wild-type and GADD45A knockout male C57BL/6J mice to 7 Gy of X rays and 24 h later RNA was isolated from whole blood and subjected to whole genome microarray and gene ontology analyses. Dose reconstruction analysis using a gene signature trained on gene expression data from irradiated wild-type male mice showed accurate reconstruction of either a 0 Gy or 7 Gy dose with root mean square error of ± 1.05 Gy (R^2 = 1.00) in GADD45A knockout mice. Gene ontology analysis revealed that irradiation of both wild-type and GADD45A-null mice led to a significant overrepresentation of pathways associated with morbidity and mortality, as well as organismal cell death. However, based on their z-score, these pathways were predicted to be more significantly overrepresented in GADD45A-null mice, implying that GADD45A deletion may exacerbate the deleterious effects of radiation on blood cells. Numerous immune cell functions and quantities were predicted to be underrepresented in both genotypes; however, differentially expressed genes from irradiated GADD45A knockout mice predicted an increased deterioration in the numbers of T lymphocytes, as well as myeloid cells, compared with wild-type mice. Furthermore, an overrepresentation of genes associated with radiation-induced hematological malignancies was associated with GADD45A knockout mice, whereas hematopoietic and progenitor cell functions were predicted to be downregulated in irradiated GADD45A knockout mice. In conclusion, despite the significant differences in gene expression between wild-type and GADD45A knockout mice, it is still feasible to identify a panel of genes that could accurately distinguish between irradiated and control mice, irrespective of pre-existing inflammation status.

FIG. 1.

Differentially expressed genes. Panel A: Significantly differentially expressed genes in wild-type and GADD45A knockout (KO) mouse blood after 7 Gy X-rays relative to unirradiated mice and under basal conditions comparing the two genotypes. Total number and percentage of upregulated and downregulated of significantly differentially expressed genes in WT and KO mouse blood (p < 0.001) on day 1 following 7 Gy X-rays relative to unirradiated mice and under basal conditions comparing KO versus WT mice. Panel B: Venn diagram showing overlap of genes that are differentially expressed in response to 7 Gy X-rays. Panel C: Impact of GADD45A on the fold-change of radiation response signature genes. Fold changes (FC) in differentially expressed genes from irradiated mice of GADD45A KO mice compared with radiation fold changes (FC) of differentially expressed genes from irradiated wild-type mice. R2 values represent the fit of the gene fold changes to a line. P values were calculated using the paired Student’s t-test. WT: wildtype; KO: GADD45A knockout.

FIG. 2.

Diseases and functions significantly over- or under-represented in wild-type and GADD45A knockout mice identified by IPA. Panel A: Top 20 biofunctions commonly differentially regulated in both genotypes. Panel B: Top-20 functions overrepresented in wild-type irradiated mice. Panel C: Top-20 biofunctions overrepresented in GADD45A knockout mice. Panel D: Stem cell-related diseases and functions significantly over- or under-represented in wild-type and GADD45A-null. Functions displaying an absolute ∣z∣ score greater than 2.000 (marked by a dotted line) and showing Benjamini-corrected p value < 0.05 were considered significant. WT: wild-type; KO: GADD45A knockout; ir: irradiated; ui: unirradiated.

FIG. 3.

Death- (Panel A) and phagocytosis- (Panel B) related functions in wild-type, p38DKI, p38DN, and GADD45A KO mice exposed to 7 Gy X-rays. Functions displaying an absolute ∣z∣ score greater than 2.000 (marked by a dotted line) and showing Benjamini-corrected p value < 0.05 were considered significant. WT: wild-type; KO: GADD45A knockout; p38αβ^Y323F: p38 MAPK double knockin; p38α^AF/+: p38MAPK dominant negative; ir: irradiated; ui: unirradiated.

FIG. 4.

Cancer-related functions. Enriched cancer-related functions in irradiated wild-type and GADD45A knockout mouse blood compared with control (unirradiated) animals. A p value < 0.05 and an absolute ∣z∣ score 2.000 (marked by the dotted lines) were considered significant. WT: wild-type; KO: GADD45A knockout; ir: irradiated; ui: unirradiated.