Metabolic phenotyping reveals a lipid mediator response to ionizing radiation

Abstract

Exposure to ionizing radiation has dramatically increased in modern society, raising serious health concerns. The molecular response to ionizing radiation, however, is still not completely understood. Here, we screened mouse serum for metabolic alterations following an acute exposure to γ radiation using a multiplatform mass-spectrometry-based strategy. A global, molecular profiling revealed that mouse serum undergoes a series of significant molecular alterations following radiation exposure. We identified and quantified bioactive metabolites belonging to key biochemical pathways and low-abundance, oxygenated, polyunsaturated fatty acids (PUFAs) in the two groups of animals. Exposure to γ radiation induced a significant increase in the serum levels of ether phosphatidylcholines (PCs) while decreasing the levels of diacyl PCs carrying PUFAs. In exposed mice, levels of pro-inflammatory, oxygenated metabolites of arachidonic acid increased, whereas levels of anti-inflammatory metabolites of omega-3 PUFAs decreased. Our results indicate a specific serum lipidomic biosignature that could be utilized as an indicator of radiation exposure and as novel target for therapeutic intervention. Monitoring such a molecular response to radiation exposure might have implications not only for radiation pathology but also for countermeasures and personalized medicine.

Figure 1

Study design and workflow for the metabolic phenotyping. CB57Bl/6 mice were irradiated (8 Gy; n = 5) or subjected to the same treatment minus irradiation (sham control group; n = 5). Blood was collected, and serum samples were prepared and divided into two aliquots. Before extraction, a mixture of internal standards was added to the serum to normalize for variations in sample preparation or MS detection. Initial discovery data were further investigated using complementary multiplexed metabolic-profiling approaches. The results were integrated for the generation of a unique metabolic biosignature, which indicates exposure to radiation.

Figure 2

Targeted metabolic profiling. (A) PLS-DA analysis showed a marked separation of serum metabolites belonging to irradiated and sham control mice, highlighting the features that contributed most to the variance between the two groups. (B) Important features identified by PLS-DA. The colored boxes on the right indicate the relative concentrations of the corresponding metabolite in each group under study. The variable importance in projection (VIP), a weighted sum of squares of the PLS loadings, takes into account the amount of explained Y variation in each dimension. (C, D) Levels of diacyl PCs (C) and ether PCs (D) in irradiated and sham control (n = 5, Student’s t test; ***, p < 0.001). The data represent the mean ± SEM.

Figure 3

Targeted oxylipin profiling. (A) PLS-DA analysis showed a marked separation of serum oxylipins belonging to irradiated and sham control mice, highlighting the features that contributed most to the variance between the two groups. (B) Important features identified by PLS-DA. The colored boxes on the right indicate the relative concentrations of the corresponding metabolite in each group under study. Variable importance in projection (VIP), a weighted sum of squares of the PLS loadings, takes into account the amount of explained Y variation in each dimension. (C, D) Levels of omega-6 oxylipins (C) and omega-3 oxylipins (D) in irradiated and sham control mice (n = 5, Student’s t test; *, p < 0.05; ***, p < 0.001). The data represent the mean ± SEM.

Figure 4

Lipidomic biosignature of irradiated mice. (A) Correlation analysis was used to visualize the overall relationships between different features and the irradiated phenotype. (B) Clustering result shown as a heatmap (distance measure using Pearson; clustering algorithm using Ward) providing an intuitive visualization of the characteristic lipidomic biosignature found in irradiated mice versus sham control mice. Each colored cell on the map corresponds to a concentration value, with samples in rows and features/compounds in columns. Displayed are the top 25 lipids, ranked by Student’s t tests.

Figure 5

Pathway analysis. Diet-derived omega-6 linoleic acid (LA) and omega-3 alpha-linolenic acid (ALA) are transformed into longer chains PUFAs by the sequential action of desaturases and elongases. PUFAs can be found in blood as unesterified fatty acids, esterified to phospholipids, or converted into the oxygenated metabolites oxylipins. (A, B) The activities of COX, LOX, and CYP450 enzymes catalyze the formation of hundreds of oxylipins species with different biological activities starting from the omega-6 PUFAs precursors (A) and the omega-3 PUFAs (B). The irradiated mice had marked alterations in the LOX, COX, and CYP450 pathways, resulting in the increase of omega-6 oxylipins (red) and decrease of omega-3 oxylipins (green).