Host microbiome depletion attenuates biofluid metabolite responses following radiation exposure

Development of novel biodosimetry assays and medical countermeasures is needed to obtain a level of radiation preparedness in the event of malicious or accidental mass exposures to ionizing radiation (IR). For biodosimetry, metabolic profiling with mass spectrometry (MS) platforms has identified several small molecules in easily accessible biofluids that are promising for dose reconstruction. As our microbiome has profound effects on biofluid metabolite composition, it is of interest how variation in the host microbiome may affect metabolomics based biodosimetry. Here, we 'knocked out' the microbiome of male and female C57BL/6 mice (Abx mice) using antibiotics and then irradiated (0, 3, or 8 Gy) them to determine the role of the host microbiome on biofluid radiation signatures (1 and 3 d urine, 3 d serum). Biofluid metabolite levels were compared to a sham and irradiated group of mice with a normal microbiome (Abx-con mice). To compare post-irradiation effects in urine, we calculated the Spearman's correlation coefficients of metabolite levels with radiation dose. For selected metabolites of interest, we performed more detailed analyses using linear mixed effect models to determine the effects of radiation dose, time, and microbiome depletion. Serum metabolite levels were compared using an ANOVA. Several metabolites were affected after antibiotic administration in the tryptophan and amino acid pathways, sterol hormone, xenobiotic and bile acid pathways (urine) and lipid metabolism (serum), with a post-irradiation attenuative effect observed for Abx mice. In urine, dose×time interactions were supported for a defined radiation metabolite panel (carnitine, hexosamine-valine-isoleucine [Hex-V-I], creatine, citric acid, and Nε,Nε,Nε-trimethyllysine [TML]) and dose for N1-acetylspermidine, which also provided excellent (AUROC ≥ 0.90) to good (AUROC ≥ 0.80) sensitivity and specificity according to the area under the receiver operator characteristic curve (AUROC) analysis. In serum, a panel consisting of carnitine, citric acid, lysophosphatidylcholine (LysoPC) (14:0), LysoPC (20:3), and LysoPC (22:5) also gave excellent to good sensitivity and specificity for identifying post-irradiated individuals at 3 d. Although the microbiome affected the basal levels and/or post-irradiation levels of these metabolites, their utility in dose reconstruction irrespective of microbiome status is encouraging for the use of metabolomics as a novel biodosimetry assay.