Molecular Mechanisms of Radiation Induced Dna Damage: H-Addition to Bases, Direct Ionization and Double Strand Break

Structures and properties of C.(-H) and of TH. were obtained from quantum mechanical calculations. New AMBER parameters for these radicals were obtained to fit their structures and charge distributions. Molecular mechanics simulations of the conformational changes induced in a 12-mer of DNA, d(CGCGAATTCGCG), by these radicals show that the distances between the base and the C₂' of the sugar becomes shorter. Such changes suggest that the base radical can abstract the H,' and transfer the radical from the base to the sugar. Once the radical becomes centered on the sugar a strand break can follow. A simultaneous formation of guanine and thymine radicals on opposite strands may lead to a double strand break.     

Electron spin resonance study of DNA irradiated with an argon-ion beam: evidence for formation of sugar phosphate backbone radicals

In this study, the effects of high-LET radiation on DNA were investigated and compared with the effects of gamma radiation. Hydrated DNA samples at 77 K were irradiated with argon-ion beams ((36)Ar or (40)Ar beam at energies between 60 and 100 MeV/nucleon). The individual free radicals formed were identified and their yields were investigated by electron spin resonance spectroscopy. Argon-ion irradiation resulted in lower yields of base ion radicals and higher yields of neutral radicals than gamma irradiation. A hitherto unknown species was assigned to the radical formed by C-O bond rupture at the deoxyribose C3', resulting in a sugar carbon-centered radical. A previously characterized phosphorus-centered radical was also found. The formation of each of these species was accompanied by an immediate strand break. G values, k values, and analyses for the individual yields of neutral radicals and ion radical composition for argon-ion-irradiated hydrated DNA are reported and compared to those found previously for gamma-irradiated DNA. The lower G values and k values for ion radicals and the higher fraction of neutral radicals found for argon-ion-irradiated DNA are attributed to differences in track structure inherent in the two radiations.     

Hydroxyl radical-induced strand break formation of poly(U) in anoxic solution. Effect of dithiothreitol and tetranitromethane

The role of dithiothreitol (DTT) and tetranitromethane (TNM) on the yields of radiation-induced strand break formation in polyuridylic acid (poly(U] was studied in anoxic aqueous solutions at neutral pH by low-angle laser light-scattering. From G (single-strand breaks) as a function of DTT concentration it follows that two different processes lead to OH radical-induced single-strand break (ssb) formation. Only one of the two processes, which accounts for 80 per cent of the ssb formation, is inhibited by DTT, the other one, 20 per cent, is not inhibited. The 'repair' process is attributed to H-donation to the C-6-yl radical of the uracil moiety. The C-6-yl radical is produced by OH addition to the C-5 position of the uracil moiety. It follows that the sugar radicals, in contrast to earlier suggestions, do not seem to be repaired by DTT at the low concentrations used. The strand break formation not inhibited by DTT is induced by radicals other than the uracil-6-yl radical, e.g. the uracil-5-yl or the OH radicals reacting with the sugar moiety. The strong reduction of G(ssb) from 2.3 to 0.2 on addition of TNM is also discussed.     

Yields of radiation-induced main chain scission of poly U in aqueous solution: strand break formation via base radicals

The G values for single-strand breaks G(ssb) in polyuridylic acid (poly U) have been measured by low-angle laser light scattering in aqueous solutions under various conditions (e.g. in the presence of N2O, Ar and t-butanol). In N2O-saturated solutions at room temperature and pH 5.6, the G(ssb) is 2.3. The efficiency of ssb formation was found to be 41 per cent for OH radicals, 19 per cent for H atoms and congruent to zero for e-aq. On the basis of 20 per cent and less than 5 per cent attack on the sugar moiety by OH radicals and H atoms, respectively, the large G(ssb) values obtained cannot be explained solely as resulting from radicals produced by reaction of OH radicals and H atoms on the sugar moiety. It is therefore proposed that base radicals produced by the reaction of OH radicals or H atoms with the uracil moiety can also lead to chain break formation in poly U via radical transfer to the sugar moiety.     

Sugar radicals in DNA: isolation of neutral radicals in gamma-irradiated DNA by hole and electron scavenging

In this investigation of the radical formation and the reaction of radicals in gamma-irradiated DNA, we report the isolation of putative neutral radicals by the scavenging of holes by Fe(CN)6(4-) and of electrons by Fe(CN)6(3-). Experiments are performed under conditions that emphasize direct and quasi-direct effects (collectively called direct-type effects.) Samples containing Fe(CN)6(4-) show effective scavenging of holes and the ESR spectra obtained arise principally from DNA anion radicals and neutral radicals. On the other hand, for samples containing Fe(CN)6(3-), electron scavenging is highly efficient, and the resulting spectra arise principally from guanine cation radicals and neutral radicals. When both Fe(CN)6(4-) and Fe(CN)6(3-) are present, a near complete scavenging of cation radicals and anion radicals is observed at 77 K, and the ESR spectra that result originate predominantly with neutral radicals which are assigned predominantly to radicals on the sugar phosphate backbone. A notable finding is the presence of spectral components that indicate the formation, through the rupture of the C3'-O bond, of a neutral deoxyribose radical; a concurrent strand break must accompany formation of this radical. This radical was previously reported in argon-ion-irradiated DNA and now, for the first time, is reported in DNA irradiated with low-LET radiation.     

Strand cleavage of supercoiled DNA by water-soluble peroxyl radicals. The overlooked importance of peroxyl radical charge

It is well established that the peroxyl radicals formed during the thermal decomposition of 2,2'-azobis(amidinopropane), ABAP, in oxygenated water can cleave double-stranded DNA, from which fact it has been concluded that peroxyl radicals, as a general class, can induce DNA strand scission. However, the ABAP-derived radicals are positively charged, and DNA is a negatively charged polyanion. Moreover, the relatively small and, therefore, free to diffuse peroxyl radicals likely to be formed in vivo will generally be negatively charged or neutral. Plasmid supercoiled DNA [pBR 322, 4361 base pairs (bp)] was reacted with known, equal fluxes of two positively charged peroxyl radicals, a negatively charged peroxyl radical, and a neutral peroxyl radical. The two positively charged peroxyl radicals degraded >/=80% of the supercoiled pBR 322 at a flux of 4 radicals/bp, but the negatively charged and neutral peroxyl radicals had no significant effect even at a flux as high as 24 radicals/bp. The same lack of effect on the DNA was also observed with high fluxes of superoxide/hydroperoxyl radicals. Similar results were obtained with another supercoiled DNA, pUC 19, except that pUC 19 is somewhat more sensitive to strand scission by positively charged peroxyl radicals than pBR 322. We conclude that most of the peroxyl radicals likely to be formed in vivo have little or no ability to induce DNA strand scission and that the potential role of electrostatics in radical/DNA reactions should always be considered.     

The role of hydroxyl radicals in tetrachlorohydroquinone induced DNA strand break formation in PM2 DNA and human fibroblasts

Tetrachlorohydroquinone (TCHQ), which has previously been identified as a metabolite of pentachlorophenol, induces DNA strand breaks in isolated DNA and in human fibroblasts. Strand break formation in PM2 DNA is prevented by the addition of catalase and the hydroxyl radical scavengers DMSO, ethanol and mannitol, whereas addition of SOD reduced SSB only slightly. Oxygen radicals are formed by the autoxidation of TCHQ to the tetrachlorosemiquinone radical. Desferrioxamine (0.2 mM) completely abolished strand break formation, whereas the metal chelator DETAPAC (1 mM) reduced SSB by only 8.5%. The formation of the semiquinone radical at physiological conditions is shown by ESR spectroscopy. Exposure of human fibroblasts to TCHQ also leads to DNA single strand breaks measured by the alkaline elution assay. These were reduced by addition of 5% DMSO. This indicates that at least part of the strand break formation in human cells is also due to the action of hydroxyl radicals.     

OH-induced free radicals in 3'-UMP and poly(U): spin-trapping and radical chromatography

Characterization of OH-induced free radicals using 3'-UMP and poly(U) was performed by a method combining spin-trapping and radical chromatography. A N2O-saturated aqueous solution containing 3'-UMP and 2-methyl-2-nitrosopropane as a spin-trap was X-irradiated. The spin adducts generated by the reactions of OH radicals with 3'-UMP were separated by paired-ion HPLC and the separated spin adducts were identified by ESR spectroscopy. In the case of poly(U), the spin adducts were digested to oligonucleotides with RNase A and then separated and identified in the same manner as 3'-UMP. The free radicals observed for poly(U) were identical to those for 3'-UMP. The 5-yl radical and the 6-yl radical were identified as precursors of various oxidized products of the base moiety, and the 4'-yl radical and 5'-yl radical, formed by H-abstraction at the C-4' and C-5' positions of the sugar moieties, respectively, were identified as precursors of strand breaks. The 1'-yl radical, produced by H-abstraction at the C-1' position of the sugar moiety, was also identified. From the similarity of the free radicals of 3'-UMP and poly(U), it is suggested that the reactivities of OH radicals with nucleotides are identical to those in polynucleotides.     

Mapping of peroxyl radical induced damage on genomic DNA

We have examined the DNA damage produced by reaction of peroxyl radicals with human fibroblast DNA. DNA damage consisted of both strand breaks and base modifications. The extent of strand breaks and base modifications induced as a function of peroxyl radical concentration was determined by quantitation of fragment size distributions using denaturing glyoxal-agarose gel electrophoresis. Both strand breaks and base modifications increased in a log linear fashion with respect to peroxyl radical concentration. Oxidative base modifications were observed to occur to a greater extent than strand breaks at every concentration measured. The sequence-specific distribution of peroxyl radical induced base damage was mapped for 803 nucleotide positions using the method of ligation mediated PCR. A total of 87% of all guanine positions in the examined sequences was found to be significantly oxidized. The order of reactivity of DNA bases toward oxidation by peroxyl radicals was found to be G > C > T. Adenine is essentially unreactive. The yield of oxidative base modifications at guanines and cytosines by peroxyl radicals depends on the exact specification of 5' and 3' flanking bases in a polarity dependent manner. Every guanine in the 5'XGC3' motif was found to be oxidized, where X is any 5' neighbor. In contrast, 5' and 3' purine flanks drastically reduced the extent of peroxyl radical G oxidation. The pattern of base modification and the influence of nearest neighbors differs substantially from that previously reported for hydrogen peroxide damage mediated by low valent transition metal ions for the identical DNA sequences.     

Cigarette smoke-induced DNA damage in cultured human lung cells: role of hydroxyl radicals and endonuclease activation.

Cigarette smoke can cause DNA single strand breaks in cultured human lung cells (T. Nakayama et al., Nature, 314 (1985) 462-464) but the mechanisms behind this DNA damage have not been clearly elucidated. In the present study we have investigated the possibility that one of the major constituents in cigarette smoke, hydroquinone, may be important for mediating smoke-induced DNA damage in the human epithelial lung cell line, A 549, and the mechanisms behind this damage. Cells were exposed to cigarette smoke, hydrogen peroxide, or hydroquinone, in the absence and presence of different inhibitors, and the resulting DNA damage was assessed either as DNA single strand break formation or formation of the oxidative DNA adduct, 8-hydroxydeoxyguanosine. It was found that (i) exposure to cigarette smoke, hydrogen peroxide or hydroquinone causes a rapid decrease in the intracellular thiol level and a considerable DNA single strand break formation, (ii) the formation of DNA single strand breaks in cells exposed to cigarette smoke is inhibited by catalase, dimethylthiourea, and o-phenantroline, suggesting that hydroxyl radicals generated from iron-catalyzed hydrogen peroxide dissociation are involved in the DNA damage, (iii) hydroquinone causes considerable DNA strand break formation that is blocked by aurintricarboxylic acid, an inhibitor of endonuclease activation, and by BAPTA, an intracellular calcium chelator, (iv) addition of hydroquinone to a smoke condensate greatly enhances its ability to cause DNA single strand breaks, and (v) smoke, but not hydroquinone, causes formation of 8-hydroxydeoxyguanosine, a DNA damage product induced by the action of hydroxyl radicals on the DNA base, deoxyguanosine. These findings suggest that the ability of cigarette smoke to cause DNA single strand breaks in cultured lung cells is due to mechanisms involving hydroxyl radical attack on DNA and endonuclease activation. They also suggest that hydroquinone is an important contributor to the DNA damaging effect of cigarette smoke on human lung cells.     

Esr Spectra of Radicals of Single-Stranded and Double-Stranded DNA in Aqueous Solution. Implications for Oh-Induced Strand Breakage

In situ photolysis at 20^oC (argon plasma light source, $, $ 200 mm) of oxygen-free solutions containing 2mM H₂0₂ and heat-denatured, single-stranded (sS)DNA from calf-thymus resulted in the ESR spectra of the 6-hydroxy-5,6-dihydro-thymin-5-yl {1} and 5-methyleneuracil {3} radicals linked to the sugar-phosphate backbone. They were generated by reaction of OH radicals with DNA. By comparison of the decay characteristics of the ESR signals with rate constants from pulse-conductivity measurements [E. Bothe, G.A. Qureshi and D. Schulte-Frohlinde, Z. Naturforsch. 38c 1030, (1983)] the thymine-derived radicals {1} and {3} can be excluded as precursors of the fast, dominating component of strand breakage of ssDNA. In the absence of H₂0₂ from native, doubie-stranded (ds)DNA an ESR signal was obtained (singlet, g ～ 2.004, $1/2 ～ 0.8 mT) which was assigned to the deprotonated guanine radical cation, {G'(-H)} of a DNA subunit. It is assumed that by the UV irradiation the guanine radical cation, {G⁺}, is generated, either by monophotonic photoionisation or by electron transfer to pyrimidine bases. By rapid transfer of the bridging proton from {G⁺} to the hydrogen bonded cytosine {G'(-H)} is formed. When photolysis of dsDNA was carried out in the presence of H₂0₂, reaction of photolytically generated OH resulted in peroxyl radicals and purine radicals. The oxygen for formation of the peroxyl radicals is probably produced by reaction of {G' (-H)} with H₂0₂. Photolysis of N₂0-saturated solutions containing dsDNA or ssDNA provided another possibility of generation of OH radicals. Under those conditions the OH-induced radicals {1} and {3} were obtained not only from ssDNA but also from dsDNA.     

On the chemical yield of base lesions, strand breaks, and clustered damage generated in plasmid DNA by the direct effect of X rays

The purpose of this study was to determine the yield of DNA base damages, deoxyribose damage, and clustered lesions due to the direct effects of ionizing radiation and to compare these with the yield of DNA trapped radicals measured previously in the same pUC18 plasmid. The plasmids were prepared as films hydrated in the range 2.5 < Gamma < 22.5 mol water/mol nucleotide. Single-strand breaks (SSBs) and double-strand breaks (DSBs) were detected by agarose gel electrophoresis. Specific types of base lesions were converted into SSBs and DSBs using the base-excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The yield of base damage detected by this method displayed a strikingly different dependence on the level of hydration (Gamma) compared with that for the yield of DNA trapped radicals; the former decreased by 3.2 times as Gamma was varied from 2.5 to 22.5 and the later increased by 2.4 times over the same range. To explain this divergence, we propose that SSB yields produced in plasmid DNA by the direct effect cannot be analyzed properly with a Poisson process that assumes an average of one strand break per plasmid and neglects the possibility of a single track producing multiple SSBs within a plasmid. The yields of DSBs, on the other hand, are consistent with changes in free radical trapping as a function of hydration. Consequently, the composition of these clusters could be quantified. Deoxyribose damage on each of the two opposing strands occurs with a yield of 3.5 +/- 0.5 nmol/J for fully hydrated pUC18, comparable to the yield of 4.1 +/- 0.9 nmol/J for DSBs derived from opposed damages in which at least one of the sites is a damaged base.     

Evidence for the formation of strand-break precursors in hydroxy-attacked thymidine 5'-monophosphate by the spin trapping method

A method combining spin trapping, ESR, and HPLC was employed to obtain evidence for the formation of sugar radicals in OH-attacked TMP with special emphasis on the detection of strand-break precursors of DNA. OH radicals were produced by irradiating an N2O-saturated aqueous solution with X-rays. When an N2O-saturated aqueous solution containing TMP and a spin trapping reagent, MNP, was irradiated with X-rays, it was estimated on the basis of theoretical calculations using rate constants that 94% of the TMP radicals were induced by OH radicals. Since several spin adducts between TMP radicals and MNP, as well as the byproducts of the spin trapping reagent itself, were produced, reverse-phase HPLC was used to separate them. The presence of six spin adducts was confirmed by ESR examination. Further examination of these spin adducts by UV absorbance spectrophotometry showed the presence of a chromophore at 260 nm in three adducts. Since a gradual increase in the release of unaltered base from these adducts was observed when they were allowed to stand for 0-22 h at room temperature, they could be regarded as the spin adducts of sugar radicals and MNP. ESR spectra from the spin adducts were consistent with hydrogen abstraction radicals at the C1', C4', and C5' positions of the sugar moiety. These radicals appeared to be precursors of AP sites and strand breaks. In addition to these spin adducts, ESR spectra that were consistent with the spin adducts of base radicals (the C5 and C6 radicals) and MNP were observed.     

Unaltered free base release from d(CGCGCG)2 produced by the direct effect of ionizing radiation at 4 K and room temperature

Unaltered free base release in d(CGCGCG)2 exposed to X rays at 4 K or room temperature was measured by HPLC. Samples were prepared either as films hydrated to a level of Gamma = 2.5 mol water/mol nucleotide or as polycrystalline with Gamma approximately 7.5 mol water/mol nucleotide. X irradiation of films at 4 K, followed by annealing to room temperature, resulted in yields for cytosine and guanine of G(Cyt) = 0.036 +/- 0.001 micromol/J and G(Gua) = 0.090 +/- 0.002 micromol/J. Irradiation of films at room temperature gave similar yields. The yields for polycrystalline d(CGCGCG)2 X-irradiated at room temperature were G(Cyt) = 0.035 +/- 0.005 micromol/J and G(Gua) = 0.077 +/- 0.023 micromol/J. The total free base release yield, G(fbr), was 0.124 +/- 0.008 micromol/J for films and 0.112 +/- 0.028 micromol/J for polycrystalline samples. G(fbr) is believed to be a good estimate of total strand break yield. The yields of total free radicals trapped [G(Sigmafr)] by the d(CGCGCG)2 films at 4 K were measured by EPR. The measured value, G(Sigmafr) = 0.450 +/- 0.005 micromol/J, was used to calculate the yield of trappable sugar radicals, giving G(sugar)(fr) = 0.04-0.07 micromol/J. We found that (1) guanine release exceeded cytosine release by more than twofold, (2) G(sugar)(fr) cannot account for more than half of the free base release, and (3) G(fbr), G(Cyt) and G(Gua) were independent of the sample temperature during irradiation. Finding (1) suggests that base and or sequence influences sugar damage, and finding (2) is consistent with our working hypothesis that an important pathway to strand break formation entails two one-electron oxidations at the same sugar site.     

The biological implications of damage to DNA incorporating an 8-oxodeoxyguanine:cytosine basepair

DNA damage produced by free radicals is probably the most frequent lesion encountered by cells (Wallace, S.S., Environmental and Molecular Mutagenesis 12, 431-477, 1988 (1)). One of the most common effects is the formation of 7-hydro-8-oxodeoxyguanine due to oxygen radicals interacting with the normal guanine base. Such chemical changes appear to be important in mutagenesis, cancer and aging. We have used computer simulation techniques to model the effect of inclusion of such a modified base within a duplex strand of DNA. We find that such modifications can be stabilized within a normal sequence. The conformation of the modified base relative to the sugar residue depends on many local interactions not accessible to the isolated nucleoside. We have also studied the essential dynamics of both normal and modified sequences and show that there are only subtle changes to the dynamics on inclusion of such a modification.     

Temperature responses of dark respiration in relation to leaf sugar concentration

Changes in leaf sugar concentrations are a possible mechanism of short‐term adaptation to temperature changes, with natural fluctuations in sugar concentrations in the field expected to modify the heat sensitivity of respiration. We studied temperature‐response curves of leaf dark respiration in the temperate tree Populus tremula (L.) in relation to leaf sugar concentration (1) under natural conditions or (2) leaves with artificially enhanced sugar concentration. Temperature‐response curves were obtained by increasing the leaf temperature at a rate of 1°C min^?1. We demonstrate that respiration, similarly to chlorophyll fluorescence, has a break‐point at high temperature, where respiration starts to increase with a faster rate. The average break‐point temperature (T_RD) was 48.6 ± 0.7°C at natural sugar concentration. Pulse‐chase experiments with ¹⁴CO₂ demonstrated that substrates of respiration were derived mainly from the products of starch degradation. Starch degradation exhibited a similar temperature‐response curve as respiration with a break‐point at high temperatures. Acceleration of starch breakdown may be one of the reasons for the observed high‐temperature rise in respiration. We also demonstrate that enhanced leaf sugar concentrations or enhanced osmotic potential may protect leaf cells from heat stress, i.e. higher sugar concentrations significantly modify the temperature‐response curve of respiration, abolishing the fast increase of respiration. Sugars or enhanced osmotic potential may non‐specifically protect respiratory membranes or may block the high‐temperature increase in starch degradation and consumption in respiratory processes, thus eliminating the break‐points in temperature curves of respiration in sugar‐fed leaves.     

Chemical probing of RNA with the hydroxyl radical at single-atom resolution

While hydroxyl radical cleavage is widely used to map RNA tertiary structure, lack of mechanistic understanding of strand break formation limits the degree of structural insight that can be obtained from this experiment. Here, we determine how individual ribose hydrogens of sarcin/ricin loop RNA participate in strand cleavage. We find that substituting deuterium for hydrogen at a ribose 5′-carbon produces a kinetic isotope effect on cleavage; the major cleavage product is an RNA strand terminated by a 5′-aldehyde. We conclude that hydroxyl radical abstracts a 5′-hydrogen atom, leading to RNA strand cleavage. We used this approach to obtain structural information for a GUA base triple, a common tertiary structural feature of RNA. Cleavage at U exhibits a large 5′ deuterium kinetic isotope effect, a potential signature of a base triple. Others had noted a ribose-phosphate hydrogen bond involving the G 2′-OH and the U phosphate of the GUA triple, and suggested that this hydrogen bond contributes to backbone rigidity. Substituting deoxyguanosine for G, to eliminate this hydrogen bond, results in a substantial decrease in cleavage at G and U of the triple. We conclude that this hydrogen bond is a linchpin of backbone structure around the triple.     

Direct detection of radical generation in rat liver nuclei on treatment with tumour-promoting hydroperoxides and related compounds

EPR spin trapping has been employed to directly detect radical production in isolated rat nuclei on exposure to a variety of hydroperoxides and related compounds which are known, or suspect, tumour promoters. The hydroperoxides, in the absence of reducing equivalents, undergo oxidative cleavage, generating peroxyl radicals. In the presence of NADPH (and to a lesser extent NADH) reductive cleavage of the OO bond generates alkoxyl radicals. These radicals undergo subsequent rearrangements and reactions (dependent on the structure of the alkoxyl radical), generating carbon-centred radicals. Acyl peroxides and peracids appear to undergo only reductive cleavage of the OO bond. With peracids this cleavage can generate aryl carboxyl (RCO₂·) or hydroxyl radicals (HO·); with acyl peroxides, aryl carboxyl radicals are formed and, in the case of t-butyl peroxybenzoate, alkoxyl radicals (RO·). The radicals detected with each peroxide are similar in type to those detected in the rat liver microsomal fraction, although the extent of radical production is lower. The subsequent reactions of the initially generated radicals are similar to those determined in homogenous chemical systems, suggesting that they are in free solution. Experiments with NADPH/NADH, heat denaturation of the nuclei and various inhibitors suggest that radical generation is an enzymatic process catalysed by haemproteins, in particular cytochrome P-450, and that NADPH/cytochrome P-450 reductase is involved in the reductive cleavage of the OO bond. The generation of these radicals by the rat liver nuclear fraction is potentially highly damaging for the cell due to the proximity of the generating source to DNA. Several previous studies have shown that some of the radicals detected in this study, such as aryl carboxyl and aryl radicals, can damage DNA, via various reactions which results in the generation of strand breaks and adducts to DNA bases: these processes are suggested to play an important role in the tumour promoting activity of these hydroperoxides and related compounds.     

Spontaneous Cleavage of Single and Double Stranded 4′-DNA Radicals Under Aerobic Conditions

Abstract

The O₂-induced strand scission of 4′-DNA radicals is initiated by a reversible O₂ addition reaction. The rate coefficient of the O₂ release from the 4′-DNA peroxyl radical is 1.00 s^?1 in single strands and 0.05 s^?1 in double strands at 20°C. Because of this reversibility, an O₂-dependent strand cleavage occurs only in the presence of H-donors which trap the 4′-DNA peroxyl radicals yielding DNA hydroperoxides. At very low H-donor concentrations the strand scission is the result of an O₂-independent, spontaneous reaction even under aerobic conditions.     

ESR studies on DNA cleavage induced by enediyne C-1027 chromophore

C-1027 belongs to the family of chromoprotein antitumor antibiotics, which contain a carrier apoprotein and a highly unstable enediyne chromophore. The enediyne spontaneously aromatizes to generate p-benzyne biradical, and subsequently abstracts hydrogens from the DNA sugar backbone, resulting in cleavage of the double strand. Using spin-trapping methods, we obtained direct proof of radical intermediates during an DNA cleavage, and found intriguing difference in behavior between the trapping agents 2-methyl-2-nitrosopropane (MNP) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO): MNP added to the sugar radicals of the DNA, whereas DMPO directly trapped a phenyl radical or p-benzyne biradical derived from the C-1027 chromophore.