Analysis by UPLC-TOFMS of 24-h urine samples collected immediately after exposure of mice to 3 and 8 Gy γ radiation produced data matrices of m/z compared to retention time compared to normalized ion intensity that, when subjected to multivariate data analysis by OPLS, revealed distinct metabolomic phenotypes for each dose and for sham-irradiated animals (). From the top 22 ions contributing to this clustering and inter-phenotype separation, a number of urinary biomarkers were unequivocally identified using tandem mass spectrometric comparison with authentic standards. A novel biological molecule, 3-hydroxy-2-methylbenzoic acid 3-O-sulfate was a biomarker of 3 Gy but not 8 Gy. N-Hexanoylglycine and β-thymidine were biomarkers of exposure to both 3 and 8 Gy ( and ). Taurine was a biomarker of 8 Gy only. In general, the increase in biomarker excretion in urine of exposed over sham-irradiated animals was 1.2- to 2.5-fold. The one exception was β-thymidine, in which case the presence in urine was elevated six- to seven-fold after γ irradiation. In addition, several in-source fragment and isotope ions were identified. Finally, we demonstrate a clear dose–response relationship in the global view of the urine metabolite profile visualized using GEDI.
The chemical identities of four markers have been elucidated, of which two, β-thymidine and N-hexanoylglycine, are validated and quantified across the two experiments. Because these ions are elevated in urine from exposed animals at both doses, we conclude that these are specific biomarkers of radiation exposure. In addition, we found 3-hydroxy-2-methylbenzoic acid O-sulfate statistically significantly elevated in the urine of animals exposed to 3 Gy but not 8 Gy compared with controls. We also observed that taurine is statistically significantly elevated in the urine of mice exposed to 8 Gy but not 3 Gy compared with controls. The point estimate of mean taurine level in the urine from mice exposed to 3 Gy is elevated over that of the controls, albeit not significantly. This is suggestive of a dose–response relationship that needs to be examined further at doses intermediate between 3 and 8 Gy. In addition, there are several other ions among the 22 ions highlighted in each experiment that are not common to both experiments.
Although it was not possible to identify unambiguously all urinary constituents that were elevated after γ irradiation of mice, a bioinformatic technique was used that demonstrated that a large number of urinary constituents co-varied across the sample set with the aforementioned biomarkers. In other words, the phenotypes for 3 Gy and 8 Gy that were seen as distinct clusters in the plots of OPLS scores (, respectively) arose due to numerous differences in urinary constituents between the irradiated and sham-irradiated animals. This can be seen from the GEDI self-organizing maps, where groups of nine interconnected tiles, which represented hundreds of both negative () and positive () ions, increased () in intensity in a dose-dependent manner, while others decreased (), also in a clear dose-dependent fashion. This is an important proof of principle of radiation metabolomics and is also the first time that GEDI self-organizing maps have been used to analyze and display global in vivo metabolomic data. We take these observations to be a sign of the existence of a rich source of additional biomarkers of radiation exposure.
Using pairs of biomarkers (), it may ultimately be possible to predict whether a mouse has been exposed to γ radiation and perhaps also the general dose range. This approach, refined by the future addition of biomarkers, is expected to lead to a metabolomics-based protocol for non-invasive radiation biodosimetry in humans. Considerably more work needs to be done, and a re-evaluation of the published literature appears to be in order. To this end, lists the small molecule biomarkers of ionizing radiation exposure reported for both laboratory animals and humans, together with the calculated m/z values of their protonated and deprotonated molecular ions. It is of note that none of the validated radiation biomarkers that are reported here have been reported previously. The published biomarkers fall into the classes of neurotransmitter metabolites, excised DNA adducts, reactive oxygen products, and general metabolic intermediates. A search for these positive and negative ions in our data set established that none of these published biomarkers were statistically significantly elevated within 1 day after exposure of mice to 3 and 8 Gy γ radiation except for 192.027, citric acid. In this case, we attempted to but could not determine whether citric acid or isocitric acid or both were detected. Thus the identity of the [M-H]− ion of m/z 191.0192 elevated in urine from radiation-exposed mice remains to be elucidated. Future research will determine whether any of the ions listed in are elevated at later times.
Historical Biomarkers of Radiation Exposure
The question arises as to the metabolic origins of the novel radiation biomarkers reported here. The most dramatic change after irradiation was in β-thymidine (), which may reflect increased synthesis, decreased use or elevated renal tubular outward transport. It is also possible that the products of oxidative DNA damage, thymine glycol and thymidine glycol (19
), might be metabolically reconverted to thymidine, although this is known not to occur in E. coli
) and is therefore unlikely. Interestingly, when [3
H]thymidine was administered intravenously to patients, the radioactivity found in urine was approximately 100 times that in plasma (42
), suggesting that extracellular thymidine is rapidly excreted into urine. The elevated thymidine excretion reported here is therefore a potential marker of increased DNA breakdown and cell turnover due to γ radiation.
Elevated urinary excretion of N
-hexanoylglycine is usually interpreted as a sign of impaired medium-chain fatty acid metabolism, that is, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, although this is usually accompanied by the excretion of dicarboxylic acids and free fatty acids (43
). These additional metabolic signs did not appear in our metabolomic analysis, suggesting that the elevated appearance of N
-hexanoylglycine in urine may not be a result of an effect of γ radiation on hepatic mitochondrial MCAD. We have recently reported that urinary excretion of N
-hexanoylglycine is reduced 20-fold after activation of the nuclear receptor PPARα in mice (44
) and it has been reported that PPARα appears to play a role in the response of mice to 10 Gy γ radiation (45
). How these two lines of evidence are related is currently unknown.
Elevated taurine excretion in urine was first reported to be associated with carbon tetrachloride liver damage (46
), but metabolomic studies have since characterized it as a general urinary marker of hepatotoxicity (47
). Since taurine is an end product of cysteine catabolism, it has been proposed that urinary excretion of taurine represents evidence of increased cysteine use in the liver in response to toxic injury (48
). The elevation in urinary excretion of taurine reported here is modest and occurred only after the 8-Gy dose (). Increased hepatic or renal cysteine/glutathione turnover is one possible explanation.
The elevation of urinary 3-hydroxy-2-methylbenzoic acid 3-O-sulfate after 3 Gy but not 8 Gy is without precedent. All possible isomers of this compound were synthesized in situ and evaluated by tandem mass spectrometry, and this organic acid sulfate gave a perfect match to the urinary peak by both retention time and mass fragmentography (). To our knowledge, the parent 3-hydroxy-2-methylbenzoic acid has not been described before in biological systems. Isomeric hydroxymethylbenzoic acids, however, are known bacterial metabolites and may arise from the gut flora. Further characterization of this biomarker falls beyond the scope of this report.
In summary, we report here a metabolomic investigation of mice after γ irradiation with 3 and 8 Gy. OPLS analysis of mass spectrometric data matrices revealed novel biomarkers that were statistically significantly elevated in urine. GEDI self-organizing maps demonstrate the existence of dose-dependent excretion of a subset of global urinary biomarkers. These data will be useful to help design strategies for noninvasive radiation biodosimetry through metabolomics in human populations.