The effect of low dose rate on metabolomic response to radiation in mice.

Metabolomics has been shown to have utility in assessing responses to exposure by ionizing radiation (IR) in easily accessible biofluids such as urine. Most studies to date from our laboratory and others have employed γ-irradiation at relatively high dose rates (HDR), but many environmental exposure scenarios will probably be at relatively low dose rates (LDR). There are well-documented differences in the biologic responses to LDR compared to HDR, so an important question is to assess LDR effects at the metabolomics level. Our study took advantage of a modern mass spectrometry approach in exploring the effects of dose rate on the urinary excretion levels of metabolites 2 days after IR in mice. A wide variety of statistical tools were employed to further focus on metabolites, which showed responses to LDR IR exposure (0.00309 Gy/min) distinguishable from those of HDR. From a total of 709 detected spectral features, more than 100 were determined to be statistically significant when comparing urine from mice irradiated with 1.1 or 4.45 Gy to that of sham-irradiated mice 2 days post-exposure. The results of this study show that LDR and HDR exposures perturb many of the same pathways such as TCA cycle and fatty acid metabolism, which also have been implicated in our previous IR studies. However, it is important to note that dose rate did affect the levels of particular metabolites. Differences in urinary excretion levels of such metabolites could potentially be used to assess an individual's exposure in a radiobiological event and thus would have utility for both triage and injury assessment.

Dynamics and stability of individual base pairs in two homologous RNA-DNA hybrids.

Nuclear magnetic resonance spectroscopy and proton exchange have been used to characterize two RNA-DNA hybrids from the tR2 intrinsic transcription terminator site of phage lambda. The hybrids have the same base sequence [5'-GGCGCAGGCC(T/U)(T/U)CC-3'/5'-GGAAGGCC(T/U)GCGCC-3'] but differ from each other by an interchange of DNA and RNA strands. The opening of single base pairs in the two hybrids is characterized by measuring the rates of exchange of imino protons with solvent protons as a function of the concentration of a proton acceptor (ammonia base) at 10 degrees C. The free energy change in the opening reaction provides a measure of the stability of the base pair, while the rates of opening and closing define the base pair dynamics. The results demonstrate that, within the same base sequence context, dA-rU base pairs are less stable than dT-rA base pairs. The differences in stability are enhanced when two dA-rU base pairs are located next to each other in the hybrid structure. For the G-C base pairs, the rates of opening and closing and the stability are affected by the base sequence context and by the nature of the sugar moiety attached to the guanine. The dominant feature of the base sequence is the proximity of the dA-rU base pair, which destabilizes the G-C base pair when the guanine is located on the DNA strand. Two G-C base pairs (namely, those in the fourth and 10th positions) exhibit large differences in their opening and closing rates between the two hybrids, while maintaining the same stability. These results provide the first demonstration that, for RNA-DNA hybrid structures with the same base sequence, the opening dynamics and the stability of individual base pairs are strongly influenced by the chemical nature of each strand.

Identification of urinary biomarkers from X-irradiated mice using NMR spectroscopy.

In a major radiological event, rapid screening of radiation-exposed individuals for possible medical intervention is critical. Here we suggest a high-throughput, non-invasive approach to identify radiation biomarkers in urine and demonstrate a proof of principle in mice. Mice were whole-body irradiated (8 Gy X rays), and urine samples were collected from both irradiated and control mice for 7 days after exposure. (1)H nuclear magnetic resonance (NMR) spectra of all the urine samples were acquired on a spectrometer operating at a proton frequency of 600 MHz. The multivariate data were analyzed by principal component analysis (PCA). The resulting biomarkers revealed a broad range of metabolism changes, including creatine, succinate, methylamine, citrate, 2-oxoglutarate, taurine, N-methyl-nicotinamide, hippurate and choline. The temporal dependence of several biomarkers on radiation exposure was also explored. Combining several metabolomic biomarkers with different temporal dependence could provide an estimate of when the radiation exposure occurred. These results will be helpful in projecting metabolomic "fingerprints" in humans exposed to radiation.

Structural energetics and base-pair opening dynamics in sarcin-ricin domain RNA.

The sarcin-ricin domain is a universal element of the RNA from the large ribosomal subunit. The domain is part of the binding site for elongation factors and is specifically cleaved by the toxins alpha-sarcin and ricin. In this work, we have mapped the energetics and dynamics of individual structural motifs in a 29-mer RNA oligomer containing the sarcin-ricin domain. The stability of individual base pairs in the structure was characterized from measurements of the exchange rates of imino protons using nuclear magnetic resonance spectroscopy at 10 degrees C. The measurements also provided the rates of opening and closing for selected base pairs. The results reveal that the structural stabilization free energies in the sarcin-ricin domain are broadly distributed between 2.9 and 10.6 kcal/mol. One of the least stable sites in the structure is the noncanonical G-A base pair located next to the phosphodiester bond that is cleaved by alpha-sarcin. The low stability of this base pair supports the proposal that cleavage by alpha-sarcin occurs by a base flipping mechanism. The opening dynamics of other base pairs is affected by elements of the structure such as the bulged-G motif and its cross-strand stacking. Participation in these motifs increases the lifetimes of the bases in an open, solvent-accessible conformation.

Sequence-dependence of the energetics of opening of at basepairs in DNA.

Proton exchange and nuclear magnetic resonance spectroscopy are being used to characterize the energetics of opening of AT/TA basepairs in the DNA dodecamer 5'-d(GCTATAAAAGGG)-3'/5'-d(CCCTTTTATAGC)-3'. The dodecamer contains the TATA box of the adenovirus major late promoter. The equilibrium constants for opening of each basepair are measured from the dependence of the exchange rates of imino protons on ammonia concentration. The enthalpy, entropy, and free energy changes in the opening reaction of each basepair are determined from the temperature dependence of the exchange rates. The results reveal that the opening enthalpy changes encompass a wide range of values, namely, from 17 to 29 kcal/mol. The largest values are observed for the AT basepairs in 7th and 8th positions. These values, and the exchange rates of the corresponding imino protons, suggest that these two basepairs open in a single concerted reaction. The enthalpy changes for opening of the central six basepairs are correlated to the opening entropy changes. This enthalpy-entropy compensation minimizes the variations in the opening free energies among these central basepairs. Deviations from the enthalpy-entropy compensation pattern are observed for basepairs located close to the ends of the duplex structure, suggesting a different mode of opening for these basepairs.