Effect of Age and Sex on Gene Expression-Based Radiation Biodosimetry Using Mouse Peripheral Blood

Abstract

Blood-based gene expression profiles that can reconstruct radiation exposure are being developed as a practical approach to radiation biodosimetry. However, age and sex could potentially limit the accuracy of the approach. In this study, we determined the impact of age on the peripheral blood cell gene expression profile of female mice exposed to radiation and identified differences and similarities with a previously obtained transcriptomic signature of male mice. Young (2 months) and old (24 months) female mice were irradiated with 4 Gy X-rays, total RNA was isolated from blood 24 hours later and subjected to whole-genome microarray analysis. Dose reconstruction analyses using a gene signature trained on gene expression data from irradiated young male mice showed accurate reconstruction of 0 or 4 Gy doses with root mean square error of ±0.75 Gy (R2 = 0.90) in young female mice. Although dose reconstruction for irradiated old female mice was less accurate than young female mice, the deviation from the actual radiation dose was not statistically significant. Pathway analysis of differentially expressed genes revealed that after irradiation, apoptosis-related functions were overrepresented, whereas functions related to quantities of various immune cell subtypes were underrepresented, among differentially expressed genes from young female mice, but not older animals. Furthermore, young mice significantly upregulated genes involved in phagocytosis, a process that eliminates apoptotic cells and preserves tissue homeostasis. Both functions were also overrepresented in young, but not old, male mice following 4 Gy X-irradiation. Lastly, functions associated with neutrophil activation that is essential for killing invading pathogens and regulating the inflammatory response were predicted to be uniquely enriched in young but not old female mice. This work supports the concept that peripheral blood gene expression profiles can be identified in mice that accurately predict physical radiation dose exposure irrespective of age and sex.

Keywords: Age; Gene expression; Mice; Radiation biodosimetry; Sex.

Fig. 1.

Total white blood cell count (WBC) and percentages of different blood types in young and old female mice 1 day after 4 Gy X-irradiation. (a) total WBC counts, (b) lymphocytes (%), (c) neutrophils (%), (d) monocytes (%), (e) platelets counts. Data represent the mean S.E.M (n = 3). P values were calculated using the unpaired Student’s t-test. ns: not significant (p ≥ 0.05). YUI, young unirradiated; YIR, young irradiated; OUI, old unirradiated; OIR, old irradiated.

Fig. 2.

Differentially expressed genes. (a) Significantly differentially expressed genes in young and old female mouse blood (p < 0.005) following 4 Gy X-rays (X-ray) relative to unirradiated mice and comparing unirradiated old versus unirradiated young female mice (control). Percent of upregulated (up) and downregulated (down) genes are shown. (b) Venn diagram showing overlap of differentially expressed genes in irradiated young male (green) and female (blue) versus irradiated old male (red) and female (yellow) mice (c) Heatmaps of combined effects of age and sex on commonly differentially expressed genes in young female and young male mice exposed to radiation.

Fig. 3.

Impact of age and sex on the fold-change of radiation response signature genes. Fold changes (FC) in differentially expressed genes from irradiated mice of different age and sex compared with radiation fold changes (FC) of differentially expressed genes from irradiated young male 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. MY: male young; FY: female young; MO: male old; FO: female old.

Fig. 4.

Immune cell death-related functions are overrepresented among genes responding to radiation in irradiated young female mice (blue). A muted trend of response was also observed for old female mice (red). Dotted line marks absolute z-score = 2.0.

Fig. 5.

Phagocytosis-associated biological functions are overrepresented among genes responding to radiation in young mice both for (a) female mice and (b) both sexes combined. A more muted response is seen in the old animals (red) compared with the young animals (blue). Dotted red line marks z-score = 2.0.

Fig. 6.

Neutrophil-related functions are enriched in young female mice exposed to radiation. (a) Significantly overrepresented neutrophil recruitment and activation functions. (b) Reactive oxygen generation and metabolism-related pathways in irradiated mice of different age and sex. Dotted red line marks z-score = 2.0.