Release of 5-methylene-2-furanone (5-MF), a characteristic marker of DNA deoxyribose oxidative damage at the C1′ position, was observed in significant quantities from X-irradiated DNA. This observation, which held for DNA irradiated either in aqueous solution or as a film, requires postirradiation treatment at 90° C in the presence of polyamines and divalent metal cations at biological pH. The 5-MF product was quantified by using reverse-phase HPLC. The radiation chemical yield of 5-MF comprised more than 30% of the yield of total unaltered base release. Polylysine, spermine and Be(II) showed the strongest catalytic effect on 5-MF release, while Zn(II), Cu(II), Ni(II), putrescine and Mg(II) were substantially less efficient. We have hypothesized that the 5-MF release from irradiated DNA occurs through catalytic decomposition of the 2′ -deoxyribonolactone (dL) precursor through two consecutive β -and δ -phosphate elimination reactions. A stepwise character of the process was indicated by the S-shaped time course of 5-MF accumulation. If dL proves to be the precursor to 5-MF formation, it would then follow that dL is a very important lesion generated in DNA by ionizing radiation.

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 radiation chemical yields of unaltered base release have been measured in three crystalline double-stranded DNA oligomers after X irradiation at 4 K. The yields of released bases are between 10 and 20% of the total free radical yields measured at 4 K. Using these numbers, we estimate that the yield of DNA strand breaks due to the direct effect is about 0.1 μmol J −1 . The damage responsible for base release is independent of the base type (C, G, A or T) and is not scavenged by anthracycline drugs intercalated in the DNA. For these reasons, reactions initiated by the hydroxyl radical have been ruled out as the source of base release. Since the intercalated anthracycline scavenges electrons and holes completely but does not inhibit base release, the possibility for damage transfer from the bases to the sugars can also be ruled out. The results are consistent with a model in which primary radical cations formed directly on the sugar-phosphate backbone react by two competing pathways: deprotonation, which localizes the damage on the sugar, and hole tunneling, which transfers the damage to the base stack. Quantitative estimates indicate that these two processes are approximately equally efficient.

Radiation damages DNA through two distinct types of reaction pathways, direct-type and indirect-type. 1 The indirect-type pathways entail reactions of water radiolysis products with DNA, for example, the formation of strand breaks by hydroxyl radical attack, which has been extensively studied. 2 Direct-type effects are caused by direct ionization of DNA or by transfer of holes and electrons to DNA from the surrounding hydration waters. 3-5 The direct component may constitute a significant fraction of the total radiation damage to DNA in cells where water radiolysis products are effectively scavenged by the medium. 6 Much less is known about the direct-type mechanisms and related quantitative characteristics, especially in regards to strand break formation. It is known that electron attachment to DNA is not a significant source of strand breaks. 7 Sugar radicals generated through one-electron oxidation of the sugarphosphate moiety has been implicated as the main source of strand scission from direct-type damage. 7This study reports the first direct measurement of radiation-chemical yields of individual strand break products induced by the direct effect in DNA. Two DNA oligonucleotides, d(CGCG) 2 (1) and d(CGCGCG) 2 (2), duplexes in crystalline form, were X-irradiated at two different temperatures, 4 and 293 K. HPLC in combination with photodiode array detection was used to identify and measure the chemical yields of all cleavage fragments that retained at least one unaltered base. Product identifications are based on comparison with the retention times and optical absorption spectra of authentic compounds and are supported by MALDI-TOF measurements. 8 Crystals of oligonucleotides offer the advantage of exact knowledge of the helical packing and conformation. Both 1 9 and 2 10 are in Z-conformation, and the hydration levels are relatively low (7.1 and 7.2 waters per nucleotide, respectively), 4 well below the level required for detectable production of OH radicals from the waters of hydration. 5 Previous work has shown that trapped hydroxyl radicals are not detected at 4 K in crystals of either 1 or 2. 4 Indirect-type reactions, therefore, cannot be a significant source of strand breaks in these samples.A typical HPLC chromatogram of irradiated 2 is shown in Figure 1 along with the reference and zero dose chromatograms. Chromatograms of the irradiated 1 differ from Figure 1 by the absence of peaks corresponding to end-phosphorylated tetramers and pentamers, and by the parent-compound retention time. From comparison of the crystal and reference spectra, all major damage products found in the crystal are represented in the reference sample and vice versa. The fragmentation products are identified as free bases, 3′-, and 5′-end phosphorylated strand fragments. The production of other degradation products not present in the reference sample is minimal. For example, there is no evidence of 3′-phosphoglycolates that are the products of the H4′-abstraction from DNA in the presence of oxygen (see ref 12 for a review). The...

The question of how NA base sequence influences the yield of DNA strand breaks produced by the direct effect of ionizing radiation was investigated in a series of oligodeoxynucleotides of the form (d(CG) n ) 2 and (d(GC) n ) 2 . The yields of free base release from X-irradiated DNA films containing 2.5 waters/nucleotide were measured by HPLC as a function of oligomer length. For (d(CG) n ) 2 , the ratio of the Gua yield to Cyt yield, R, was relatively constant at 2.4-2.5 for n = 2-4 and it decreased to 1.2 as n increased from 5 to 10. When Gua was moved to the 5′ end, for example going from d (CG) 5 to d(GC) 5 , R dropped from 1.9 ± 0.1 to 1.1 ± 0.1. These effects are poorly described if the chemistry at the oligomer ends is assumed to be independent of the remainder of the oligomer. A mathematical model incorporating charge transfer through the base stack was derived to explain these effects. In addition, EPR was used to measure the yield of trapped-deoxyribose radicals at 4 K following X-irradiation at 4 K. The yield of free base release was substantially greater, by 50-100 nmol/J, than the yield of trapped-deoxyribose radicals. Therefore, a large fraction of free base release stems from a nonradical intermediate. For this intermediate, a deoxyribose carbocation formed by two one-electron oxidations is proposed. This reaction pathway requires that the hole (electron loss site) transfers through the base stack and, upon encountering a deoxyribose hole, oxidizes that site to form a deoxyribose carbocation. This reaction mechanism provides a consistent way of explaining both the absence of trapped radical intermediates and the unusual dependence of free base release on oligomer length.