Stochastic model of free radical yields in oriented DNA exposed to densely ionizing radiation at 77K

Monte Carlo simulation techniques were used to calculate the probability that thymine radical anions (T.-), formed by the slowing-down of high-energy protons in oriented DNA, will undergo a secondary protonation reaction. By assuming a large asymmetry in the thermal conductivity of oriented DNA fibres we predict a significant enhancement of protonation of T.- when the proton flux is incident on the sample parallel to the orientation of the DNA. These results are in qualitative agreement with experimental data on the production of TH. radicals when oriented DNA is exposed to fast neutrons.     

Free-radical formation in gamma-irradiated oriented DNA containing electron-affinic radiosensitizers.

Electron paramagnetic resonance (e.p.r.) was used to study the free radicals induced by gamma-irradiation at 77 K in oriented DNA with incorporated electronaffinic radiosensitizing compounds (4-nitroacetophenone, metronidazole, and Ro-07-0582). The observed e.p.r. spectra were compared with those obtained from pure oriented DNA, which had previously been analysed in detail and found to consist mainly of two components, arising from anion redicals on thymine, and cation radicals on guanine. The major spectral changes caused by the radiosensitizers could be explained as a considerable increase in the formation of cationic free radicals on guanine. There were also indications of the formation of anion radicals on the radiosensitizer molecules. No hydrogen-addition free radicals on thymine were observed when the radiosensitized samples were annealed, in contrast to the pure DNA samples.     

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.     

The effects of ionizing radiation on DNA: the role of thiols as radioprotectors

Electron loss from N-(2-mercaptopropionyl) glycine (PSH) gave an EPR detectable radical anion, PS-.SP(-). When the PSH derivative was frozen in aqueous DNA solutions to 77 K and exposed to ionizing radiation, normal damage to the DNA was detected by EPR spectroscopy. However, on annealing above 77 K, central EPR features for the DNA base radical cations and anions gave central features assigned to PS-.SP(-) sigma*-radical anions, together with outer features for 5-6-dihydro-5-thymyl radicals, TH.. It is proposed that on freezing, the PSH molecules are constrained into a glassy region around the DNA, and that, on annealing, electron donation gives PS. radicals, with loss of quanine radical-cations, G(.+). The PS. radicals were not detectable, but on reaction with another PSH molecule, gave good EPR spectra for PS-.SP(-) radical-anions. These results indicate that PSH had little effect on the yield of the other base radicals C(.-)/T(.-). Also, growth of TH. radicals, formed from protonated thymine radical-anions, T(.-), were detected. We conclude that the primary effect of PSH is to capture the G(.+) centers, and thus could either prevent or repair radiation damage to DNA.     

Ionic base radicals in gamma-irradiated oriented non-deuterated and fully deuterated DNA.

The free radicals induced by gamma-irradiation at 77 K in non-deuterated and fully deuterated oriented samples of moist algal DNA have been studied by electron paramagnetic resonance (e.p.r.). The e.p.r. spectra from the non-deuterated sample were found to be similar to those from calf-thymus DNA studied previously. Numerical spectra simulations were performed based on the previously proposed interpretation of the DNA spectra at 77 K in terms of a mixture of two spectral components arising from anionic and cationic base radicals (probably on thymine and guanine, respectively). The simulations were found to account satisfactorily for the main characteristics of the e.p.r. spectra from both the non-deuterated and the fully deuterated algal DNA sample.     

ESR and optical absorption evidence for free radical involvement in the photosensitizing action of furocoumarin derivatives and for their singlet oxygen production.

Frozen aqueous solutions of thymine and its derivatives were irradiated with visible light (lambda greater than 320 nm) in the presence of various furocoumarins. ESR analysis revealed the induction of hydrogen adduct free radicals at C-6 position of thymine, only with those furocoumarin derivatives which show a skin-photosensitizing ability. It has been shown, moreover, that the photocycloaddition of psoralen to thymine, which is responsible for the biological effects of this dye, is inhibited when the induction of free radicals in thymine moiety has been prevented by electron scavengers. It is suggested that the free radicals observed could be involved in the biological photosensitization. The mechanism of free radical generation and singlet oxygen production by furoccoumarins were also investigated.     

Electron-spin-resonance studies of a chlorpromazine derivative bound to DNA fibres.

Solutions of DNA, spin-labelled with the radical cation of chlorpromazine, were used to produce oriented species of fibres pulled from a gel obtained by ultracentrifugation. The electron spin resonance spectra, recorded at X and Q band frequencies, are given for both gel and fibres; 14N hyperfine coupling parameters were obtained by computer fitting. The spectra are explained in terms of a strongly immobilized label having one principal hyperfine tensor axis parallel to the axis of the DNA helix, and the preferential orientation of the chlorpromazine ions with their planes perpendicular to the DNA helical axis.     

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.     

Structure of a RecA-DNA complex from linear dichroism and small-angle neutron-scattering in flow-oriented solution

Small-angle neutron-scattering (SANS) and ultraviolet linear dichroism (l.d.) were measured on identical samples of a RecA-double-stranded (ds) DNA complex, including cofactor adenosine 5'-O-thiotriphosphate, which were aligned by flow in two equivalent Couette devices made of niobium and silica, transparent to neutrons and to ultraviolet light, respectively. The SANS anisotropy indicates a modest orientation of the RecA-dsDNA fiber with the helix axis parallel to the flow field. By correlation with the corresponding l.d. of the DNA at the same orientation conditions, it is inferred that the DNA bases have a local orientation that is approximately perpendicular to the helix axis. By comparison with the worse orientation in single-stranded DNA-RecA, this conclusion suggests that the dsDNA in its complex with RecA is not strand separated, and may be accommodated as an essentially unperturbed, straight double helix running along the RecA polymer fiber. The SANS anisotropy is also found to support the assignment of a subsidiary intensity maximum as originating from the pitch of a helical fiber.     

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.     

Comparison of radiolysis products of thymine and thymidine with e.s.r. results.

Radicals determined by e.s.r. spectrometry of irradiated thymine or thymidine and radiolytic products generated under tha ction of gamma rays in aerated aqueous solutions have been compared. This comparison lies mainly in the fact that a radical R gives rapidly the corresponding peroxide ROOH. The authors have isolated and characterized twenty peroxides, i.e., the four isomers cis (-), cis (+), trans (-), trans(+) of 6-hydroperoxy-5-hydroxy-5,6-dihydrothymidine; the four isomers cis (-), cis (+), trans (-), trans (+) of 5-hydroperoxy-6-hydroxy-5,6-dihydrothymidine; 5-hydroperoxy-2-deoxyuridin;cis and trans 6-hydroperoxy-5-hydroxy-5,6-dihydrothymine; cis and trans 5-hydroperoxy-6-hydroxy-5,6-dihydrothymine; 5-hydroperoxymethyl-uracil; 5-hydroperoxy-5,6-dihydrothymine;cis and trans 6-hydroperoxy-5,6-dihydrothymine; 5-hydroperoxy-5-methyl barbituric acid; 5-hydroperoxy-5-methyl hydantoin; trans 5,6-dihydroperoxy-5,6-dihydrothymine. Most of thethymine and thymidine radicals hypothesized or described in the literature were correlated to these peroxides. However, the presence of certain peroxides could not be explained by recognized radicals. Taking advantage of this fact, the existence of new thymine or thymidine radicals so far unknown can be predicted.     

E.s.r. of spin-trapped radicals in gamma-irradiated aqueous solutions of nucleic acids and their constituents.

The gamma-radiolysis of de-aerated neutral aqueous solutions of uracil, thymine, cytosine and of the corresponding nucleosides and nucleotides and of calf-thymus DNA was investigated. For uracil and thymine, the U.V. photolysis of aqueous solutions containing H2O2 was also studied. The short-lived radicals were spin-trapped by tert-nitrosobutane and identified by electron-spin-resonance spectroscopy. For all compounds two or more radicals were observed, and these could be distinguished by following the thermal decay of the spin adducts. Radicals formed by the addition of H or OH at the C(5) or C(6) positions of the pyrimidine derivatives were observed in all cases. Sodium formate was used as a scavenger for H and OH to identify the radicals formed by eaq-. Spin-trapped radicals in gamma-irradiated aqueous solutions of polynucleotides exhibited broad e.s.r. lines. For DNA gel, additional narrow lines due to scission products were also found.     

Radiation-induced free radicals in DNA, DNA level in tissues and tissue radiosensitivity

Radiochemical yields (G-values) of H-adducts of thymine bases of DNA (TH) in frozen (77 K) gamma-irradiated mouse and rat tissues were measured. The content of DNA and the number of TH radicals formed in DNA mass of 10(12)D at a dose of 1 Gy (beta parameter) were determined for each of the studied tissue. It was shown that beta parameter, which indicated DNA in situ radiosensitivity, was different for different tissues: it was higher for radiosensitive tissues. The possible causes of the effect observed are discussed.     

ESR spectroscopy of flow-oriented cation radicals of phenothiazine derivatives and phenoxathiin intercalated in DNA.

Several derivatives of the phenothiazine cation radicals intercalated into DNA have been investigated using a new flow orientation technique. The anisotropic hyperfine coupling constants of both the parallel and the perpendicular orientation relative to the magnetic field were measured and compared to previous results, which used different techniques. The phenoxathiin cation radical could also be stabilized by intercalation into DNA at pH 4, but the orientation technique revealed no further information due to the poor resolution of the experimental spectra.     

Analysis of double-helix motions with spin-labeled probes: binding geometry and the limit of torsional elasticity

The e.p.r.⁵ spectra of a family of spin-labeled probes non-covalently bound to DNA have been measured as functions of helix orientation, packing density and temperature. The spectra are interpreted in terms of the geometrical relations between the helix axis and the orbital containing the unpaired electron and in terms of the motions of the helix. Torsional and flexural motions can be distinguished.Spectra from well-ordered helices have been obtained using fully hydrated DNA fibers that are in thermodynamic equilibrium with unbound probe in dilute salt solution. The binding equilibria are similar to the equilibria in dilute DNA solution. The spatial relations between the spin label and the helix, inferred from the spectra, correspond closely to the structure expected on the basis of intercalation perpendicular to the helix axis and a sterically hindered amide bond between the spin label and the intercalating moiety of the probe. Viscometric measurements with one probe also indicate intercalation.Linear e.p.r. spectra of solutions, randomly condensed DNA, and fibers show substantial torsional motion but no detectable flexure on the linear e.p.r. time scale (> 300 ns). The correlation time of a propidium-based probe is much longer than that of aminoacridine intercalators. The probes with short correlation times are considered to be too weakly coupled to the adjacent base-pairs to be reliable indicators of DNA dynamics. For the propidium probe the correlation time, 30 nanoseconds, and its temperature dependence are compared with the properties expected according to four models: tight rotational coupling along the entire length of the helix; swivels at fixed intervals; a two-state exchange; and elastic rotational coupling between adjacent nucleotide pairs. In terms of the fourth model, the results suggest that each nucleotide pair undergoes random oscillation with an r.m.s. amplitude of not more than 4 ° to 5 ° at room temperature. That value agrees with estimates made in other ways.     

Visible-light-induced OH radicals in DNA-proflavine complexes: an e.p.r. and spin trapping study

Using the spin-trapping technique, irradiation with visible light of complexes between DNA and proflavine was shown to generate OH radicals. The characteristic spectra were not obtained when proflavine or DNA were irradiated alone, nor when oxygen was absent. Using DMPO as a spin trap we found that the intensity of the DMPO-OH e.p.r. signal was enhanced when the molar ratio between bound proflavine and the DNA phosphorus increased up to a value of 0.15 demonstrating the efficiency of the intercalated dye molecules. A strong decrease of the e.p.r. signal was observed in the presence of various OH. scavengers like t-butanol, isopropanol or sodium benzoate. The OH. production was also decreased when the irradiation was made in the presence of SOD, ceruloplasmin or catalase and after addition of Chelex 100 resin.     

32P-postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini.

A 32-P-postlabeling assay has been developed that permits detection of several radiogenic base and sugar lesions of DNA at the femtomole level. The technique is based on the inability of DNase I and snake venom phosphodiesterase to cleave the internucleotide phosphodiester bond immediately 5' to the site of damage so that complete digestion of irradiated DNA with these nucleases and alkaline phosphatase yields lesion-bearing "dinucleoside" monophosphates. Because these fragments contain an unmodified nucleoside at the 5'-end of each molecule, they can be readily phosphorylated by T4 polynucleotide kinase and [gamma-32P]ATP and analyzed by polyacrylamide gel electrophoresis and reverse-phase HPLC. We observed a linear induction of total damage in DNA irradiated with 5-50 Gy. Virtually no damage was detected when the DNA was irradiated in solution containing 1 M DMSO, implicating hydroxyl radicals in the formation of these lesions. Evidence for the presence of thymine glycols and phosphoglycolate groups came from (i) a comparison of the radiation-induced products with those produced by OsO4 and KMnO4 and (ii) incubation of irradiated DNA with Escherichia coli endonuclease III and exonuclease III before analysis by the postlabeling procedure. This was confirmed by comigration of the radiogenic products with chemically synthesized markers. G values of 0.0022 and 0.0105 mumol J-1 were obtained for thymine glycol and phosphoglycolate production, respectively. The identity of the 5'-nucleotide of each isolated compound was obtained by nuclease P1 digestion. This analysis of nearest-neighbor bases to thymine glycols and phosphoglycolates indicated a nonrandom interaction between radiation-induced hydroxyl radicals and DNA.     

Free radical generation by ultrasound in aqueous solutions of nucleic acid bases and nucleosides: an ESR and spin-trapping study

Direct evidence for the detection of intermediate radicals of nucleic acid constituents induced by ultrasound in argon-saturated aqueous solution is presented. The method of spin trapping with 3,5-dibromo-4-nitrosobenzene sulphonate, which is a water-soluble, non-volatile, aromatic nitroso spin trap, combined with ESR, was used for the detection of sonochemically induced radicals. Spin adducts were also generated by OH radicals produced by UV photolysis of aqueous solution containing H2O2. ESR spectra observed from these photolysis experiments were identical to those after sonolysis. The ESR spectra of the spin adducts suggest that the major spin-trapped radical of thymine and thymidine was the 5-yl radical, and that of cytosine, cytidine, uracil, and uridine was the 6-yl radical. To compare the radicals induced by sonolysis and photolysis, the decay of the ESR spectra of the thymine and thymidine spin adducts was investigated. The decay curves of thymine and thymidine after sonolysis indicated biphasic decay. However, after photolysis the spin adducts from both compounds showed very little decay. These results suggest that the observed spin adducts in the sonolysis of pyrimidine bases and nucleosides were formed by OH radical and H atom addition to the 5,6 double-bond.     

Radiolysis of spin-labeled DNA: an electron spin resonance investigation

The reactions of free and DNA-bound 2,2,5,5-tetramethylpyrrolidine-N-oxyl (PROXYL) probes with radicals generated during radiolysis of dilute aqueous solutions of DNA were examined. For the free PROXYL probe in deaerated solution with each of the four nucleotides (dAMP, dCMP, dGMP, and TMP) it was found that the pyrimidine radicals were more reactive toward the probe than were the purine radicals. Reactions of the electron adduct of TMP and the hydroxyl radical adducts of dAMP, dGMP, and TMP with the probe resulted in little or no reduction of the probe. For TMP these results are consistent with the fact that both the protonated electron and hydroxyl radical adducts of TMP will covalently bind to the nitroxide function of the probe. Reduction of the PROXYL probe was observed in reactions with the hydroxyl radical adduct of dCMP and with the electron adducts of dAMP, dCMP, and dGMP. Results of the radiolysis of the free PROXYL probe in deaerated dilute solution of DNA suggest that the PROXYL probe protects the DNA from water radical attack as the ratio of DNA bases to PROXYL probe increases above 50:1. Reactions of DNA-bound probes are dependent on the depth of the nitroxide function in relation to the major groove of the DNA helix. Two probes with tether lengths which are less than the depth of the major groove show an expected increase in reactions with DNA base radicals as compared to a probe with a tether that extends beyond the groove. The longer probe is involved largely in reactions with sugar and water radicals along the periphery of the DNA helix. In the presence of oxygen, there is a dramatic decrease in the loss of both the free and DNA-bound probes due to the lack of reaction of these probes with peroxyl radicals formed by the addition of molecular oxygen to DNA radicals.     

Structure of a hydroxyl radical induced DNA-protein cross-link involving thymine and tyrosine in nucleohistone

Hydroxyl radical induced formation of a DNA-protein cross-link involving thymine and tyrosine in nucleohistone is described. Hydroxyl radicals were generated in N2O-saturated aqueous solution by ionizing radiation. Samples of nucleohistone were hydrolyzed with HCl and trimethylsilylated. Analysis of irradiated samples by gas chromatography-mass spectrometry with selected-ion monitoring showed the presence of a thymine-tyrosine cross-link on the basis of typical fragment ions from the previously known mass spectrum of its trimethylsilyl derivative. The yield of this DNA-protein cross-link in nucleohistone was measured at incrementing doses of radiation and found to be a linear function of radiation dose between 14 and 300 Gy (J.kg-1). This yield amounted to 0.003 mumol.J-1. The mechanism of formation of this DNA-protein cross-link is thought to result from H atom abstraction by hydroxyl radicals from the methyl group of thymine followed by the addition of the resultant thymine radical to the carbon 3 position of the tyrosine ring and subsequent oxidation of the adduct radical.