Dose-response curves were measured for the formation of direct-type DNA products in X-irradiated d(GCACGCGTGC)(2)prepared as dry films and as crystalline powders. Damage to deoxyribose (dRib) was assessed by HPLC measurements of strand break products containing 3' or 5' terminal phosphate and free base release. Base damage was measured using GC/ MS after acid hydrolysis and trimethylsilylation. The yield of trappable radicals was measured at 4 K by EPR of films X-irradiated at 4 K. With exception of those used for EPR, all samples were X-irradiated at room temperature. There was no measurable difference between working under oxygen or under nitrogen. The chemical yields (in units of nmol/J) for trapped radicals, free base release, 8-oxoGua, 8-oxoAde, diHUra and diHThy were G(total)(fr) = 618 +/- 60, G(fbr) = 93 +/- 8, G(8-oxoGua) = 111 +/- 62, G(8-oxoAde) = 4 +/- 3, G(diHUra) = 127 +/- 160, and G(diHThy) = 39 +/- 60, respectively. The yields were determined and the dose-response curves explained by a mechanistic model consisting of three reaction pathways: (1) trappable-radical single-track, (2) trappable-radical multiple-track, and (3) molecular. If the base content is projected from the decamer's GC:AT ratio of 4:1 to a ratio of 1:1, the percentage of the total measured damage (349 nmol/J) would partition as follows: 20 +/- 16% 8-oxoGua, 3 +/- 3% 8-oxoAde, 28 +/- 46% diHThy, 23 +/- 32% diHUra, and 27 +/- 17% dRib damage. With a cautionary note regarding large standard deviations, the projected yield of total damage is higher in CG-rich DNA because C combined with G is more prone to damage than A combined with T, the ratio of base damage to deoxyribose damage is approximately 3:1, the yield of diHUra is comparable to the yield of diHThy, and the yield of 8-oxoAde is not negligible. While the quantity and quality of the data fall short of proving the hypothesized model, the model provides an explanation for the dose-response curves of the more prevalent end products and provides a means of measuring their chemical yields, i.e., their rate of formation at zero dose. Therefore, we believe that this comprehensive analytical approach, combined with the mechanistic model, will prove important in predicting risk due to exposure to low doses and low dose rates of ionizing radiation.

The purpose of this study was to determine how free radical formation (fr) correlates with single strand break (ssb) and double strand break (dsb) formation in DNA exposed to the direct effects of ionizing radiation. Chemical yields have been determined of (i) total radicals trapped on DNA at 4 K, G(∑fr), (ii) radicals trapped on the DNA sugar, G sugar (fr), (iii) prompt single strand breaks, G prompt (ssb), (iv) total single strand breaks, G total (ssb), and (v) double strand breaks, G(dsb). These measurements make it possible, for the first time, to quantitatively test the premise that free radicals are the primary precursors to strand breaks. G(∑fr) were measured by EPR applied to films of pEC (10 810 bp) and pUC18 (2686 bp) plasmids hydrated to Γ = 22 mol of water/nucleotide and Xirradiated at 4 K. Using these same samples warmed to room temperature, strand breaks were measured by gel electrophoresis. The respective values for pEC and pUC18 were G(∑fr) = 0.71 ± 0.02 and 0.61 ± 0.01 μmol/J, G total (ssb) = 0.09 ± 0.01 and 0.14 ± 0.01 μmol/J, G(dsb) = 0.010 ± 0.001 and 0.006 ± 0.001 μmol/J, and G total (ssb)/G(dsb) ~9 and ~20. Surprisingly, G sugar (fr) ≈ 0.06 μmol/ J for pUC18 films, less than half of G total (ssb). This indicates that a significant fraction of strand breaks are derived from precursors other than trapped DNA radicals. To explain this disparity, various mechanisms were considered, including one that entails two one-electron oxidations of a single deoxyribose carbon.

The purpose of this study was to elucidate the role of hydration (Γ) in the distribution of free radical trapping in directly ionized DNA. Solid-state films of pUC18 (2686 bp) plasmids were hydrated to Γ in the range 2.5 ≤ Γ ≤ 22.5 mol water/mol nucleotide. Free radical yields, G(∑fr), measured by EPR at 4 K are seen to increase from 0.28 ± 0.01 μmol/J at Γ =2.5 to 0.63 ± 0.01 μmol/J at Γ = 22.5, respectively. Based on a semi-empirical model of the free radical trapping events that follow the initial ionizations of the DNA components, we conclude that two-thirds of the holes formed on the inner solvation shell (Γ < 10) transfer to the sugar-phosphate backbone. Likewise, of the holes produced by direct ionization of the sugar-phosphate, about one-third are trapped by deprotonation as neutral sugar-phosphate radical species, while the remaining two-thirds are found to transfer to the bases. This analysis provides the best measure to date for the probability of hole transfer (~67%) into the base stack. It can thus be predicted that the distribution of holes formed in fully hydrated DNA at 4 K will be 78% on the bases and 22% on the sugar-phosphate. Adding the radicals due to electron attachment (confined to the pyrimidine bases), the distribution of all trapped radicals will be 89% on the bases and 11% on the sugar-phosphate backbone. This prediction is supported by partitioning results obtained from the high dose-response curves fitted to the two-component model. These results not only add to our understanding of how the holes redistribute after ionization but are also central to predicting the yield and location of strand breaks in DNA exposed to the direct effects of ionizing radiation.

The present study tests the hypothesis that the majority of DNA strand breaks produced by directtype effects are due to sugar free radical precursors and that these radicals are produced by direct ionization of the sugar-phosphate backbone or by hole transfer to the sugar from tightly bound water. Well-defined crystalline DNA samples of d(CGCG) 2 , d(CGCACG:GCGTGC), d(GTGCGCAC) 2 , and d((GCACGCGTGC) 2 were irradiated at 4 K, and their free radical dose response determined from 0 to 1800 kGy. A model is proposed that effectively describes the dose response curves. It includes the following parameters: the free radical concentration at saturation C max , the free radical yields G b and G s , and the destruction constants k b and k s . The subscripts b and s refer to base-centered and sugar-centered radicals, respectively. In each of these systems, the free radical concentration exhibits a remarkable resistance to dose saturation up to at least 1500 kGy. As predicted, G b > G s , the G b /G s ratio varying between 4 and 12. Likewise, k b > k s , the k b /k s ratio varying between 28 and 81. The lower cross-section for destruction of the sugar-centered radicals is consistent with the expectation that they are relatively radiation resistant. G b /G is between 0.81 and 0.92, indicating that at low doses the bases trap out 80-90% of the total free radical population. The remaining 10-20% are located on the sugar. At high dose, a larger fraction of the radicals are trapped on the backbone as seen from the ratio C mxS /C mxB , which ranges from 3.5 to 8. This unusually late onset of dose saturation closely parallels that observed for strand break products in earlier studies. There is, therefore, a good correlation between the dose response profiles of sugar-trapped radicals and strand breaks. These observations strongly support the hypothesis that sugar radicals are precursors to the majority of strand breaks produced by the direct-type effect in DNA.

The distance of hole migration through DNA determines the degree to which radiation induced lesions are clustered. It is the degree of clustering that confers to ionizing radiation its high toxicity. The migration distance is governed by a competition between hole transfer and irreversible trapping reactions. An important type of trapping is reactions that lead to formation of deoxyribose radicals, which are precursors to free base release (fbr). Using HPLC, fbr was measured in X-irradiated films of d(CGCGCGCGCG) 2 and d(CGCGAATTCGCG) 2 as well as three genomic DNAs: M. luteus, calf thymus, and C. perfringens. The level of DNA hydration was varied from Γ = 2.5 to 22 mol waters/ mol nucleotide. The chemical yields of each base, G(base), were measured and used to calculate the modification factor, M(base). This factor compensates for differences in the GC/AT ratio, providing a measure of the degree to which a given base influences its own release. In the DNA oligomers, M (Gua) > M(Cyt), a result ascribed to the previously observed end effect in short oligomers. In the highly polymerized genomic DNA, we found that M(Cyt) > M(Gua) and that M(Thy) is consistently the smallest of the M factors. For these same DNA films, the yields of total DNA trapped radicals, G tot (fr), were measured using EPR spectroscopy. The yield of deoxyribose radicals was calculated using G dRib (fr) = ∼0.11 × G tot (fr). Comparing G dRib (fr) with total free base release, we found that only about half of the fbr is accounted for by deoxyribose radical intermediates. We conclude that for a hole on cytosine, Cyt •+ , base-to-base hole transfer competes with irreversible trapping by the deoxyribose. In the case of a hole on thymine, Thy •+ , base-to-base hole transfer competes with irreversible trapping by methyl deprotonation. Close proximity of Gua protects the deoxyribose of Cyt but sensitizes the deoxyribose of Thy.