The effects on the mouse urinary metabolome of sublethal γ irradiation were investigated. Five urinary biomarkers of radiation exposure, statistically significantly elevated between 1 and 3 Gy, were unequivocally identified by tandem mass spectrometry. These are all deaminated purine and pyrimidine derivatives: dT, dU, dX, xanthine and X. All are elevated above baseline in urine at the three doses studied, 1, 2 and 3 Gy. Furthermore, we observed reduced urinary excretion of the aminopyrimidine dC at 2 and 3 Gy. However, this ion was only partially quantified. All of the biomarkers displayed a time-dependent excretion, peaking in urine at 8–12 h but returning to baseline by 36 h. The enhanced excretion of another nine compounds in urine was also observed, based upon detection of their negative ions by UPLC-TOFMS, but the chemical identities of these metabolites remain to be elucidated.
This study involved the dimensions of both dose and time. With respect to time, it was first observed that differential excretion of the biomarkers according to exposure status occurred within the first 24 h after exposure, consistent with earlier findings (4
). The response within the first 24 h was then characterized by collecting urine over 4-h periods up to 20 h. In both experiments, where 24-h and 4-h urine samples were collected, the irradiations were conducted in the late afternoon, at 5:00 p.m. ± 1 h. Late afternoon was chosen in the second experiment to maximize the chances of having 4-h urine samples from every mouse since the mice are active and produce more urine during the dark cycle. However, the possibility cannot be ruled out that diurnal variation in urine metabolite profiles confounds the observations (30
). On the other hand, if there is confounding by diurnal variation, then it can be argued that it is probably minimal given that the maximum differences (exposed compared to control) observed for each dose within the 4-h samples are similar to those seen in the corresponding 24-h samples.
The loadings S-plots shown in suggest that there are rich pools of both ESI− and ESI+ urine ions from which to select candidate biomarkers of radiation exposure. In fact, however, the loadings that correlate well with the model classification of exposure do not always present in urine in a manner that is useful for biodosimetry. An ideal biomarker is consistently elevated or attenuated in urine from all exposed individuals, and the individual variation is reasonably small. In a matrix of UPLC-TOFMS data, there are often a substantial number of ions detected in one or only a few samples but not in all of the samples of a given class or sample set. The variance estimates of the means of these types of ions are very large. The SIMCA software nevertheless calculates a correlation score for these ions without regard to the variance estimates of the means, and if they were elevated or attenuated in only one or two samples from exposed mice, they will nonetheless correlate reasonably well in an OPLS model in such a case. A test for exposure-specific differences in means will produce a null result because the variance estimates are relatively large. We focused our attention on ions that may be more useful for biodosimetry and less so for modeling by OPLS alone and eliminated those that were not significantly different from control concentrations. In fact, more of these ions that show promise for biodosimetry applications were found in the ESI− data set than in the ESI+ data set.
Our observations raise a number of important questions regarding the effect of γ radiation on the mouse. First, the excretion of N
-hexanoylglycine: We had reported in a previous study that this urinary biomarker was elevated at doses of both 3 and 8 Gy (4
). Multivariate data analysis using OPLS () shows that N
-hexanoylglycine is by far the most abundant murine urinary biomarker of γ radiation but that it is comparatively less well correlated to the OPLS model (p(corr)P = 0.54). Thus it is presumed that this biomarker, but no other metabolite of fatty acid metabolism, results from a perturbation of hexanoyl-CoA β-oxidation, resulting in its conjugation with glycine in an alternative pathway. This biomarker was not investigated further in the present study because it was significantly elevated only at doses of 3 Gy and above, and we focused here on the effects of doses of 3 Gy and below.
A dose-dependent elevated excretion of dT, dU, dX, X and xanthine was observed in the first 24 h postirradiation, together with a putative dose-related attenuated excretion of dC. It has already been noted (see ) that the pattern of excretion of the three pyrimidine derivatives dT, dU and dC might be explained by deamination of dC to dU by cytidine deaminase and/or deoxycytidine deaminase, followed by ultimate synthesis of dT by thymidylate synthase, an enzyme reported to be induced in mouse liver by γ radiation (27
). It would appear, therefore, that production of deaminated pyrimidines is due to γ-radiation exposure. The same pattern holds true for the purine derivatives that were found to be elevated in urine. Metabolomic analysis did not reveal an enhanced urinary excretion of guanosine (G), deoxyguanosine (dG), adenosine (A) or deoxyadenosine (dA). All of these nucleosides are aminopurines. In contrast, dX, X and xanthine were all elevated in urine after γ irradiation in a dose- and time-related manner ( and ). These purines are all deaminated and their formation from G,A, dG or dA in response to γ radiation is not readily explained by simple enzyme-mediated reactions (). Thus an alternative explanation must be sought. It was reported that γ irradiation of mice results in a statistically significant two- to threefold increase in serum nitrate concentration 2.5–3.0 h postirradiation that returns to baseline after 12 h (32
). This nitrate increase is consistent with the effect in mice of sublethal γ -radiation exposure on nitric oxide (NO) synthesis in the liver, intestine, lung, kidney, brain, spleen and heart (33
), on increased hepatic nitrite concentration and peroxidative damage (34
), and on attenuated hepatic glutathione concentration (34
). There is abundant evidence therefore that γ irradiation of mice increases hepatic NO synthesis.
One important property of cellular NO is that it may autooxidize to form nitrous anhydride (N2
), which then can participate in so-called nitrosative deamination of both purines and pyrimidines in DNA in situ
). Dedon and his colleagues have reported that dC can undergo such nitrosative deamination to dU, dA to 2′-deoxyinosine (dI) and dG to dX and 2′-deoxyoxanosine (dO) (28
). Although we did not detect elevated urinary dI (C10
= 251.0780) or dO (C10
= 267.0729) in our metabolomic analyses (see ), all other observations regarding the urinary excretion of elevated concentrations of deaminated pyrimidines and purines are consistent with the hypothesis that sublethal γ irradiation of mice leads to in vivo
nitrosative deamination by N2
of DNA bases in situ
. Clearly, further investigations with agents that can ameliorate NO are warranted. Such a strategy may provide new avenues for the development of radioprotective drugs.
A relationship between ionizing radiation and the urinary excretion of dT in rats was reported in the Russian literature over 40 years ago (36
) and subsequently between ionizing radiation and dU and the pyrimidine metabolite β-aminoisobutyric acid (38
). The elevated excretion of dT in rats after subcutaneous injection of 90
Sr was somewhat modest (38–64%) and did not appear until the 5th to 9th day after injection (36
). This is clearly different from the present findings. In addition, an increased excretion of β-aminoisobutyric acid was not observed. However, in the historical Russian literature, it was reported that whole-body X irradiation with 6.5 Gy produced a transient eightfold increase in urinary dT excretion at day 1 in rats (37
), which is remarkably similar to the sevenfold increase in urinary dT reported herein after 3 Gy γ irradiation and in a previous study (4
) in the mouse. Thus dT and dU may be biomarkers of ionizing radiation exposure not only in mouse and rat but also in humans. This hypothesis awaits rigorous testing.
The classical urinary biomarkers of repaired oxidative DNA damage are 8-hydroxy-2′-deoxyguanosine (8-OH-dG) (40
) together with thymine glycol and thymidine glycol (41
). No evidence of elevated 8-OH-dG (C10
= 282.0838) or of thymine glycol (C5
= 159.0406) was uncovered (see ). However, thymidine glycol (C10
= 273.0723) may possibly correspond to unidentified ion no. 11 (). These findings suggest that 1–3 Gy of γ radiation does not simply cause oxidation of dG by ROS, leading to increased urinary excretion of 8-OH-dG, and further helps substantiate the hypothesis that the effects of these doses of ionizing radiation on the mouse occur through a specific mechanism, such as nitrosative deamination of dC and dG. Further investigations are required to provide a more detailed mechanism of DNA damage from exposure to sublethal ionizing radiation.
In this report attention is focused mainly on urinary anions that were elevated in irradiated mice. In fact, the loadings scatter plots (both ESI− and ESI+) were interrogated to determine whether any attenuated species reliably indicated radiation exposure. However, little emphasis is placed on this pool of ions for two reasons. First, eventual human biomarkers of radiation exposure are more useful if they exhibit up-regulation in association with exposure. Given that initial positive results with urine biodosimetry were validated with follow-up methodologies, it can be argued that false negatives in radiation exposure assessment are more detrimental to the objective of biodosimetry than are false positives. Second, a lower success in observing meaningful, consistent and verifiable attenuated biomarkers of radiation exposure has been attained, despite what the loadings scatter plots suggest, dC being a notable exception.
The need for noninvasive high-throughput radiation biodosimetry tools is ever more pressing and prescient. A recent report from a U.S. Congressional Commission (December 2, 2008) states in the first paragraph of its executive summary, “The Commission believes that unless the world community acts decisively and with great urgency, it is more likely than not that a weapon of mass destruction will be used in a terrorist attack somewhere in the world by the end of 2013” (42
). Even if preparedness for a nuclear or radiological terrorist attack are high, the tools available for the mass screening of at-risk individuals for radiation exposure are pitifully few. Metabolomics offers a means of screening large populations in relatively short periods, providing that (1) a robust radiation metabolomic signature with unambiguous dose–response characteristics can be developed, and (2) a portable device suitable for use in the field by first responders can be designed, built, tested and validated. These are ambitious aims but ones that must be pursued aggressively. This report represents a second step in the characterization of a radiation metabolomic signature, with emphasis on both the dose response and the time course of the response. In addition, some novel insights into the mechanisms of radiation effects at sublethal doses are beginning to emerge. These studies may also ultimately have a bearing on the development of new strategies for prophylaxis and treatment of radiation sickness.