Small Molecule Responses to Sequential Irradiation with Neutrons and Photons for Biodosimetry Applications: An Initial Assessment

Abstract

Mass casualty exposure scenarios from an improvised nuclear device are expected to be far more complex than simple photons. Based on the proximity to the explosion and potential shielding, a mixed field of neutrons and photons comprised of up to approximately 30% neutrons of the total dose is anticipated. This presents significant challenges for biodosimetry and for short-term and long-term medical treatment of exposed populations. In this study we employed untargeted metabolomic methods to develop a biosignature in urine and serum from C57BL/6 mice to address radiation quality issues. The signature was developed in males and applied to samples from female mice to identify potential sex differences. Thirteen urinary (primarily amino acids, vitamin products, nucleotides) and 18 serum biomarkers (primarily mitochondrial and fatty acid β oxidation intermediates) were selected and evaluated in samples from day 1 and day 7 postirradiation. Sham-irradiated groups (controls) were compared to an equitoxic dose (3 Gy X-ray equivalent) from X rays (1.2 Gy/min), neutrons (∼1 Gy/h), or neutrons-photons. Results showed a time-dependent increase in the efficiency of the signatures, with serum providing the highest levels of accuracy in distinguishing not only between exposed from non-exposed populations, but also between radiation quality (photon exposures vs. exposures with a neutron component) and in between neutron-photon exposures (5, 15 or 25% of neutrons in the total dose) for evaluating the neutron contribution. A group of metabolites known as acylcarnitines was only responsive in males, indicating the potential for different mechanisms of action in baseline levels and of neutron-photon responses between the two sexes. Our findings highlight the potential of metabolomics in developing biodosimetric methods to evaluate mixed exposures with high sensitivity and specificity.

FIG. 1.

Pathway enrichment based on validated metabolites in urine and serum from male animals. Urine shows enrichment in polar metabolites, such as amino acids, while serum shows enrichment in metabolites associated with energy metabolism and mitochondrial involvement.

FIG. 2.

Levels of four acylcarnitines at 7 days postirradiation. Results in male serum samples show a gradual depletion, which are statistically significantly different from controls in the groups containing a neutron component. The levels of these acylcarnitines, on the other hand, are orders lower in female samples, with no significant changes from controls, demonstrating sex-dependent changes in neutron exposures and differential mechanisms of responses. P values were calculated using one-way ANOVA (statistical significance at P < 0.05), while tick marks reflect statistical significance compared to controls. All data presented are mean ± SEM

FIG. 3.

sPLS-DA score plots based on the urinary and serum signature developed in males demonstrate the effectiveness in separating the three neutron-photon groups with time dependence. Urine data utilized a combination of 13 metabolites, while serum data utilized a combination of 18 metabolites. Separation is shown for components 1 and 2 in all plots, with the variability in each component explained in percentage.

FIG. 4.

Superimposed ROC curves using Random Forests as a classification and feature ranking method, comparing neutrons and neutron-photon exposures to X rays. Each ROC curve shows the model averaged from all cross validation runs and the numbers represent the AUCs. Panel A. Urine analysis based on a biosignature of 13 metabolites from male samples extrapolated to females. Panel B. Serum analysis based on a biosignature of 18 metabolites from male samples extrapolated to females. X-axis: Specificity (false positive rate), Y-axis: Sensitivity (true positive rate). AUC >0.8 is considered a good model; AUC >0.9 is considered an excellent model.