UPLC-ESI-QTOFMS metabolomics was used to identify novel metabolites of γ-radiation exposure in rats and to confirm established biomarkers previously identified in mice by UPLC- ESI-QTOFMS (7
) and rats by GCMS (9
). Upregulated biomarkers included dT, dU and dX, N1
-acetyltaurine, taurine and N
dT, dU and dX were previously reported as biomarkers of radiation exposure in mice (8
), and dU and dT were seen as their corresponding uracil and thymine bases in rat urine after γ irradiation by GCMS-based metabolomics analysis (a result of the derivatization process for GCMS) (9
). In mice, the excretion of dT increased 7-fold and was similarly upregulated here in rats by 6.7-fold; a 7-fold increase in rats has also been observed by Zharkov et al.
). The metabolites dU and dX were increased 4.3-fold and 2.9-fold, respectively, and increased to the same extent in mice (8
). dU, dX and dT are markers of DNA damage and can be formed by hydrolytic or enzymatic deamination of dC, 2′-deoxyguanosine (dG) and 5′-MedC, respectively. They can also be formed through interactions with ROS generated during exposure to ionizing radiation and redox reactions (11
). A previous publication reported an increase in dU in mice subjected to 1–3 Gy γ radiation (8
) and a decrease in dC. Both dC and 5′-MedC were mined for and were decreased after irradiation with pcorr values from 0.65–0.7 by OPLS-DA; however, they could not be quantified. dG was below the limit of detection by UPLC-ESI-QTOFMS. dX had the lowest concentration and relative change compared to dT and dU; therefore, it is not surprising that dG could be observed. dG is also known to form 2′-deoxyoxanosine (dO) (13
). This metabolite was mined for in the UPLC-ESI-QTOFMS data (267.0729 [M-H]−
); it had the same m/z
as dX but a different structure and would thus be expected to form different fragments when subjected to tandem MS fragmentation. 267.0729 [M-H]−
was identified and fragmented to reveal dX (confirmed against an authentic standard) and not dO. Pyrimidine bases in DNA are known to be more susceptible to hydrolytic deamination than purine bases, but purines are more prone to deamination by nitrous acid; therefore, the results here indicated that spontaneous deamination was a more prevalent route of deamination than ROS (11
). Further work must be performed to determine whether the increases in dU and dT resulted from radiation-induced enzymatic activity (cytidine deaminase and/or dC deaminase and thymidylate synthase) or were due to the actions of ROS. The repair of DNA bases is important in cells exposed to ROS, and increases in other biomarkers past day +1 are of interest because of their relationship to this process.
The excretion of taurine was seen to increase for 2 days after radiation exposure by 2.5- and 2.1-fold. Taurine has previously been observed to be elevated in mouse urine after 8 Gy irradiation by 1.2-fold and was not seen at sublethal doses (7
). Taurine is metabolized from methionine and cysteine and is involved in many physiological functions such as bile acid conjugation, osmoregulation, antioxidation and detoxication. It is generally seen as a marker of hepatotoxicity. Because exposure to radiation can increase ROS, the role of taurine here may have been protective, inhibiting ROS, or an enhancement in cysteine or glutathione turnover could have occurred (7
). Another mechanism was proposed by Dilley (15
) in which an increase in taurine urinary excretion in radiation-exposed dogs and humans was a result of radiation-induced destruction of circulating lymphocytes.
Taurine is known to regulate ion channels. The urine collected from this study was previously analyzed by GCMS as described by Lanz et al.
). Water and food consumption and ion levels were documented. A large increase in phosphate with a decrease in calcium, sodium, potassium and chloride ions was seen after radiation exposure; the latter three could be attributed to a decrease in food intake, but regulation of calcium ion channels could also play a role in this here. Another ion that was highly correlated to radiation exposure was 124.9911 [M-H]−
, which was tentatively assigned as isethionic acid but could not be resolved from taurine. Isethionic acid is the hydroxyl analogue of taurine and is formed from taurine via a sulfoacetaldehyde intermediate. It has been proposed that mammals cannot directly metabolize taurine to isethionate; instead gut microflora perform this role (16
). Therefore, it is hypothesized that isethionic acid could be a biomarker produced as a response of the gut microflora to radiation. p
-Cresol, another gut microfloral metabolite, was previously identified by GCMS analyses of urine from radiation-exposed rats (9
-Acetyltaurine was also identified as an upregulated biomarker of ionizing radiation exposure, and it is a novel mammalian metabolite. It is a derivative of taurine and has only been observed previously in orb spiders (17
). It had a high correlation to radiation exposure, with a pcorr value of 0.95 by OPLS-DA for 1–2 days postirradiation. After in-house synthesis of the N
-acetyltaurine pyridine salt, quantification was possible. N
-acetyltaurine does not derive directly from taurine because a concomitant decrease of that metabolite was not observed.
-Acetylspermidine was increased after radiation exposure and forms through reversible N
-acetylation of spermidine by spermidine/spermine N1
-acetyltransferase (SSAT). The transcription of SSAT is induced in the presence of ROS, increased polyamine levels, interleukin-1 and hepatocyte growth factor. Polyamine oxidase (PAO) can convert the acetylated compound back to putrescine, producing H2
and aldehydes, which in turn can cause apoptosis and cell damage (18
). Spermidine and N1
-acetylspermidine form from arginine via putrescine; they play vital roles in cellular processes involving nucleic acids. Other polyamines in the putrescine pathway were also mined for in the data generated by MarkerLynx, but correlation to radiation was not observed. However, a previous study with TK6 cells showed an attenuation of spermine after exposure to 1 Gy γ radiation (6
). Spermine is formed from spermidine; it is possible that the spermidine precursor in both these cases was N
-acetylated instead of undergoing conversion to spermine by spermine synthase, producing the observed decrease in spermine. A similar effect was observed in rat spleen after 3 Gy γ irradiation; decreases in putrescine, spermine and spermidine were seen for 5 days after irradiation (19
). Increases in N1
-acetylspermidine have been observed previously in rat and human colon cancers as well as in many other types of cancer (20
). It has been observed that SSAT is enhanced at the beginning of growth arrest and cell death in HeLa S3 and myeloid cell lines after X and γ irradiation (24
). The induction of SSAT could potentially regulate growth arrest that results in cell death after toxic stress and play a protective role in response to ionizing radiation.
-acetylated biomarker seen to be upregulated after exposure to ionizing radiation was N
-acetyl-D-glucosamine/galactosamine-6-sulfate, an amino sugar. N
-Acetyl-D-glucosamine-6-sulfate and N
-acetyl-D-galactosamine-6-sulfate are key components of the proteoglycans dermatan, keratan and chondroitin sulfates. Exposure to X radiation in a rabbit chondrocyte culture system revealed a change in proteoglycan synthesis and a stimulation of proteoglycan degradation (26
); in this study, an increase in N
-acetylated amino sugars could result from ionizing radiation-induced degradation. However, it was not possible here to distinguish between N
-acetyl-D-glucosamine-6-sulfate and N
-acetyl-D-galactosamine-6-sulfate; they had the same retention time and fragmentation patterns.
The downregulated metabolites included a number of dicarboxylic acids and an acyl glycine. A reduced urinary excretion of dicarboxylic acids was observed previously by GCMS in the rat urine; it was proposed that this was a result of changes in renal tubular cells (9
). These dicarboxylic acids can be completely oxidized through β-oxidation to produce succinyl-CoA, which can be used in the gluconeogenesis pathway during starvation (27
); therefore, the decrease in these dicarboxylic acids was proposed to be a result of decreased food intake after irradiation. A further study was carried out in which rats were deprived of food for 24 h and urine was collected. Metabolomic analysis of this urine showed a decrease in the metabolites downregulated after irradiation. The ions that were increased after food deprivation were also mined for the upregulated radiation markers, but they were not found.
The samples taken at day −4 showed a number of metabolites that were present only at this time: hippurate, PAG, 2,8-quinolinediol and its glucuronide. These metabolites were produced as the rats became acclimatized to the metabolic cages. Hippurate is known to be formed from the microbial breakdown of larger dietary phenolics and benzoic acids, which are then excreted as hippurate after glycine conjugation in the liver (28
). PAG is derived from the metabolism of phenylalanine also aided by microbial metabolism and subsequent conjugation of phenylacetic acid with glycine (29
). Therefore, the movement of the rats from their standard group-housed cages to individual metabolic cages resulted in a disruption of the gut microfloral metabolized compounds that started to stabilize after 24 h.
Evaluation of GCMS and UPLC-ESI-QTOFMS for Metabolic Profiling
As mentioned originally, this metabolomic study was implemented previously using GCMS (9
) to analyze the urine samples and was repeated here using UPLC-ESI-QTOFMS. This was to determine how effective the two technologies are for metabolomics analysis of rat urine after radiation exposure and to maximize metabolite recovery. Initial PCA models were constructed from the UPLC-ESI-QTOFMS data and had identical groupings and patterns as seen by GCMS. A PCA scores plot showing the GCMS data taken from Lanz et al.
) () is similar to the UPLC-ESI-QTOFMS PCA scores plots in . There are some differences, however; the UPLC-ESI-QTOFMS variables cluster together within their group classifications, while the GCMS variables are spread out across PC1 and PC2. The more distinctive grouping within class by UPLC-ESI-QTOFMS could be due to the acquisition of the data or to pre-processing methods such as peak alignment and detection; different software was used to process the UPLC-ESI-QTOFMS and GCMS data.
FIG. 9 PCA scores plots from 24 h rat urine samples analyzed by GCMS. Black squares, day −4; black triangles, day−3; black triangles, day −2; black diamonds, day −1; blue squares, day 1 sham-irradiated; red squares, day +1 irradiated (more ...)
For biomarker identification, both methods revealed upregulated nucleosides, but they were seen as their respective free bases by GCMS due to the sample processing step of derivitization. These nucleosides (dU and dT) could be confirmed in both systems; however, dX could be seen only by UPLC-ESI-QTOFMS due its low abundance. Therefore, UPLC-ESI-QTOFMS is more effective for identifying low-abundance metabolites than GCMS. There were a number of other biomarkers that were observable only by UPLC-ESI-QTOFMS compared to GCMS and vice versa. Of these were polar compounds that would not derivatize well and thus were not identified by GCMS, for example, taurine and the N-acetylated metabolites. Three metabolites, glyoxylate, threonate and p-cresol, were observed by GCMS and not by UPLC-ESI-QTOFMS. The sample processing methods for UPLC-ESI-QTOFMS may have resulted in extraction of these metabolites into the pellet, and thus they could have been lost before loading onto the column. Alternatively, derivatization for GCMS may have improved the ability to observe these metabolites.
UPLC-ESI-QTOFMS-based metabolomics allowed effective identification of nine urinary biomarkers of γ radiation in rats, including a novel mammalian metabolite. These upregulated urinary biomarkers included dT, dU and dX, N1-acetylspermidine, N-acetylglucosamine/galactosamine-6-sulfate, N-acetyltaurine, N-hexanoylglycine, taurine and tentatively isethionic acid. Some of these metabolites were also identified in the mouse (dT, dU, dX, taurine, N-hexanoylglycine) and helped to reveal cross-species biomarkers for radiation. It is now of interest to look at other mammals, in particular non-human primates or humans, to ascertain whether these metabolites are shared outside of mice and rats. Further studies include analysis of the rat tissues to look for changes in gene transcription and regulation of enzymes to relate the metabolites discovered to molecular mechanisms.