By employing a simplified metabolomics protocol that uses low-mass resolution GCMS instrumentation and open-source software for bioinformatics, we demonstrate here that biomarkers of γ irradiation of rats can be detected and validated. Up-regulated biomarkers included glyoxylic acid, threonic acid, thymine, uracil and glycerol 3-phosphate. Down-regulated biomarkers included citric acid and 2-oxoglutaric acid, together with the dicarboxylic acids adipic, pimelic, suberic and azelaic acids. In addition, urinary urea and electrolyte analysis revealed that Na+
and urea excretion all declined after irradiation, while phosphate excretion was enhanced. It is of interest to evaluate potential sources of this postirradiation metabolomic phenotype to understand better the biological processes that contribute to it. Regarding elevated urinary excretion of the pyrimidine bases thymine and uracil, we have already reported that their corresponding 2′-deoxynucleosides thymidine and 2′-deoxyuridine show increased excretion in the mouse after γ irradiation at 1–3 Gy (2
), and we have postulated that they are elevated due to nitrosative deamination of the aminopyrimidine cytidine (2
). We report here that these corresponding 2′-deoxynucleosides undergo deribosylation under the conditions of our sample preparation and derivatization and yield the pyrimidine bases. In all probability, the observed pyrimidines arose from the DNA 2′-deoxynucleosides but not from the RNA nucleosides, e.g. uridine. This would be consistent with our previous reports in the mouse (2
) and with the known composition of human urine (15
The enhanced excretion of p
-cresol is of interest in that it is a known product of protein degradation whereby tyrosine is converted to p
-cresol by the gut microbiota and then excreted in the urine (16
). In a previous study (4
), the elevated urinary excretion of a related phenolic metabolite, 3-hydroxy-2-methylbenzoic acid 3-O
-sulfate, was reported in mice exposed to 3 Gy. This novel urinary metabolite was almost certainly contributed by gut floral metabolism. Gamma irradiation of mice has been reported to alter both the composition and size of the gut microbiota (17
) and presumably, therefore, its metabolic capacity.
The increased urinary excretion of glyoxylic acid may represent evidence of lipid peroxidation of polyunsaturated fatty acids (19
). This would fit with the nitrosative deamination of cytidine to 2′-deoxyuridine hypothesis as proposed previously (2
) in that it also involves the generation of reactive oxygen species (ROS) by ionizing radiation. The other up-regulated biomarker, threonic acid, is an oxidation product of ascorbic acid via dehydroascorbic acid (20
), the latter being produced when ascorbic acid reacts with ROS. This is yet further evidence that ROS is involved in the generation of radiation metabolomic urinary biomarkers. The final biomarker identified, glycerol 3-phosphate (), should not be considered a biomarker of radiation exposure since it has been identified on the basis of a reduced urine excretion in the sham-irradiated animals with an unremarkable excretion in the irradiated animals. The reason for the decline in excretion in the sham-irradiated animals is not known.
The reduced excretion of citric acid, 2-oxoglutaric acid and the four C6–C9 dicarboxylic acids is of interest for a number of reasons. At first sight, it would appear that these compounds reflect the reduced caloric intake by the irradiated rats, but this was not substantiated by the starvation experiment (). These data may reflect changes in renal tubular cells. In rats fasted for 18–24 h, Na+
and urea excretion has been reported to be reduced (21
), although we did not confirm the urea difference in our own experiments (). Our observations () clearly go beyond the effects of starvation. It is well known that 2-oxoglutaric acid (α-ketoglutarate) is an important driver of renal organic anion inward transport and dicarboxylic acid exchange in tubular cells in a Na+
-dependent fashion (22
). The changes in Na+
excretion observed here after irradiation, plus a decline in renal tubular energy production and thus 2-oxoglutaric acid availability, may be the reasons underlying a proportion of the urinary radiation metabolomic phenotype that includes reduced excretion of dicarboxylic acids.
The reduced excretion of Ca2+ and massive increase in phosphate excretion, against a background of reduced excretion in the sham-irradiated rats (), is worthy of special comment. The high urinary ratio of Ca2+/phosphate in the sham-irradiated rats is completely reversed in the irradiated animals, suggesting that these animals have developed a high plasma Ca2+/phosphate ratio by preserving Ca2+ but excreting phosphate. This is likely to be mediated by parathyroid hormone (PTH). The mechanism of release of PTH by γ radiation is unknown.
Overall, the effect of 3 Gy γ radiation on male rats was to produce a number of verifiable up-regulated and down-regulated urinary metabolomic biomarkers. The radiation metabolomic signature included phosphate (↑), glyoxylate (↑), threonate (↑), p
-cresol (↑), thymine (↑), uracil (↑), citrate (↓), 2-oxoglutarate (↓), adipate (↓), pimelate (↓), suberate (↓), and azelaate (↓). We propose that the elevated urinary concentrations of thymine and uracil detected in this study are surrogates for elevated urinary excretion of thymidine and 2′-deoxyuridine, due to chemical deribosylation under the conditions of sample preparation employed. This means that mice and rats behave similarly with respect to the postirradiation production of deaminated 2′-deoxynucleosides, by a mechanism that has been discussed elsewhere (2
). This encourages the belief that human subjects may also react similarly to these doses of γ radiation and that metabolomics may provide a practical means of rapid noninvasive biomonitoring of exposure to ionizing radiation.
Finally, it should be pointed out that in this report we describe methods for the conduct of metabolomic studies that can yield abundant urinary biomarkers and that would, in all probability, generate biomarkers using other metabolomes, such as sweat, sebum, plasma or other biofluids. The protocol described here uses low-mass-resolution gas chromatography-mass spectrometry and open-source software to uncover a metabolomic fingerprint of radiation exposure. This same protocol would also work with simple capillary gas chromatography without a mass-selective detector, providing that the investigator had a rich supply of authentic endogenous compounds, as many laboratories do. Our initial studies in this field have employed high mass resolution orthogonal quadrupole time-of-flight mass spectrometry coupled to ultra-high-resolution chromatography (2
). This capital expense is not within the reach of all laboratories. The methods described here not only offer a cost-effective alternative but also can provide a substantial yield of biomarkers. It is hoped that this will encourage the application of metabolomic protocols in the field of radiation research.