Search

Molecular products resulting from the reaction of radiation-produced dry and aqueous electrons with DNA hydrated between 2.5 and 367 waters per nucleotide (Γ) are quantified in this work. Specifically, the yields of the 5,6-dihydropyrimidines (formed from the reaction of electrons with thymine and cytosine) and molecular hydrogen (H2) (formed in the reaction of intra-spur electrons) have been studied. We find the yields for the 5,6-dihydropyrimidines are highest (41–108 nmol/J) and the yields of hydrogen lowest (14–18 nmol/J) at low hydrations (Γ Γ = 32 . This result would suggest that the H2 produced in the irradiated hydrated DNA is derived mainly from intra-spur reactions in bulk water. However, an analysis of the H2 G-values calculated based on the dry mass of DNA (i.e., an analysis of the contribution of the water alone to the H2 yields) suggests that H2 produced in irradiated hydrated DNA is derived not only from intra-spur reactions in bulk water (44–45 nmol/J) but also from DNA (61 nmol/J). This is an interesting finding that suggests fully hydrated DNA (Γ>33) forms H2 in competition with dihydropyrimidines.

Gas chromatography/mass spectrometry (GC/MS-SIM) is an excellent technique for performing both qualitative and quantitative analysis of DNA base damage products that are formed by exposure to ionizing radiation or by the interaction of intracellular DNA with activated oxygen species. This technique commonly uses a hot formic acid hydrolysis step to degrade the DNA to individual free bases. However, due to the harsh nature of this degradation procedure, the quantitation of DNA base damage products may be adversely affected. Consequently, we examined the effects of various formic acid hydrolysis procedures on the quantitation of a number of DNA base damage products and identified several factors that can influence this quantitation. These factors included (1) the inherent acid stabilities of both the lesions and the internal standards; (2) the hydrolysis temperature; (3) the source and grade of the formic acid; and (4) the sample mass during hydrolysis. Our data also suggested that the N,O-bis (trimethylsilyl)trifluoroacetamide (BSTFA) derivatization efficiency can be adversely affected, presumably by trace contaminants either in the formic acid or from the acid activated surface of the glass derivatization vials. Where adverse effects were noted, modifications were explored in an attempt to improve the quantitation of these DNA lesions. Although experimental steps could be taken to minimize the influence of these factors on the quantitation of some base damage products, no single procedure solved the quantitation problem for all base lesions. However, a significant improvement in the quantitation was achieved if the relative molecular response factor (RMRF) values for these lesions were generated with authentic DNA base damage products that had been treated exactly like the experimental samples.

The radiosensitivities and the kinetics for removal of radiation-induced DNA damage were compared for proliferative (P) and quiescent (Q) cells of the lines 66 and 67 derived from a mouse mammary adenocarcinoma. As determined from cell survival assays, the 66 and 67 Q cells were more radiosensitive than their 66 and 67 P counterparts. The rank order of their radiosensitivity was: 67 Q greater than 66 Q greater than or equal to 67 P greater than 66 P. Induction of radiation damage in the DNA of these cells, as measured by the alkaline elution technique, was identical for 66 and 67 P and Q cells. The repair of this DNA damage was biphasic for 66 and 67 P and Q cells. The half-times for the fast and slow repair phases in 66 Q cells were identical to those previously measured in 67 Q cells. The half-times of the fast and slow repair phases in 66 P cells were also identical to those previously measured in 67 P cells. However, the half-times for the fast and slow repair phases in 66 and 67 Q cells were longer than those measured in their 66 and 67 P counterparts. The 66 cell data are consistent with our previously published hypothesis that Q cells are more radiosensitive than their corresponding P cells because they repair their radiation-induced DNA damage slower. However, our results are not consistent with hypotheses that attempt to explain the radiosensitivity differences between lines 66 and 67 solely on the basis of measurable induction and repair of DNA damage.

Little computational or experimental information is available on site-specific hydroxyl attack probabilities to DNA. In this study, an atomistic stochastic model of OH radical reactions with DNA was developed to compute relative OH attack probabilities at individual deoxyribose hydrogen atoms. A model of the self-complementary decamer duplex d(CCAACGTTGG) was created including Na(+) counter ions and the water molecules of the first hydration layer. Additionally, a method for accounting for steric hindrance from nonreacting atoms was implemented. The model was then used to calculate OH attack probabilities at the various C-H sites of the sugar moiety. Results from this computational model show that OH radicals exhibit preferential attack at different deoxyribose hydrogens, as suggested by their corresponding percentage solvent-accessible surface areas. The percentage OH attack probabilities for the deoxyribose hydrogens [1H(5')+2H(5'), H(4'), H(3'), 1H(2')+2H(2'), H(1')] were calculated as approximately 54.6%, 20.6%, 15.0%, 8.5% and 1.3%, respectively, averaged across the sequence. These results are in good agreement with the latest experimental site-specific DNA strand break data of Balasubramanian et al. [Proc. Natl. Acad. Sci. USA 95, 9738-9742 (1998)]. The data from this stochastic model suggest that steric hindrance from nonreacting atoms significantly influences site-specific hydroxyl radical attack probabilities in DNA. A number of previous DNA damage models have been based on the assumption that C(4') is the preferred site, or perhaps the only site, for OH-mediated DNA damage. However, the results of the present study are in good agreement the experimental results of Balasubramanian et al. in which OH radicals exhibit preferential initial attack at sugar hydrogen atoms in the order 1H(5')+2H(5')H(4')H(3')1H(2')+2H(2')H(1').

The induction of base damage products in γ-irradiated DNA, hydrated between 2.5 and 32.8 moles of water per mole of nucleotide (Γ), was investigated using the gas chromatography/mass spectrometry-selected ion monitoring technique. In general, the yields of the measured base damage products were found to be dependent on the extent of the hydration when the DNA was irradiated under nitrogen. At low hydrations (Γ ≤ 13), the highest yields of the measured products were found for 7,8-dihydro-8-oxo-guanine, 5,6-dihydrothymine and, to a lesser extent, 2,6-diamino-4-oxo-5-formamidopyrimidine, products which are consistent with the base radicals found in low-temperature ESR studies. At higher hydrations (Γ ≤ 13), changes in DNA conformation and an increase in the attack of bulk water radicals on DNA play a significant role in the formation of radiation-induced DNA base damage products. Additional findings in our study include: (1) the sum of the yields of the products formed from electron-loss centers is greater t...

The release of unaltered bases from irradiated DNA, hydrated between 2.5 and 32.7 mol of water per mole of nucleotide (Γ), was investigated using HPLC. The objective of this study was to elucidate the yield of the four DNA bases as a function of dose, extent of hydration, and the presence or absence of oxygen. The increase in the yield of radiation-induced free bases was linear with dose up to 90 kGy, except for the DNA with Γ = 2.5, for which the increase was linear only to 10 kGy. The yield of free bases as a function of Γ was not constant in either the absence or the presence of oxygen over the range of hydration examined. For DNA with Γ between 2.5 and 15, the yield of free bases was nearly constant under nitrogen, but decreased under oxygen. However, for DNA with Γ > 15, the yield increased rapidly under both nitrogen and oxygen. The yield of free bases was described by a model that depended on two factors: 1) a change in the DNA conformation from a mixture of the A and C conformers in vacuum-dried D...