Radioprotective effects of dimethyl sulfoxide in golden hamster embryo cells exposed to gamma rays at 77 K. II. Protection from lethal, chromosomal, and DNA damage

Golden hamster embryo cells were exposed to 137Cs gamma rays in the presence or absence of dimethyl sulfoxide at both 310 and 77 K. Dimethyl sulfoxide gave significant protection against cell killing at both 310 and 77 K. The extent of radioprotection with 1.28 M dimethyl sulfoxide at 77 K was 85-89% of the lethal effects observed in the absence of dimethyl sulfoxide at 310 K; the dose-modifying factor was 5.7. Dimethyl sulfoxide also exerted protected against gamma-ray-induced DNA single-strand breaks and chromosomal aberrations with a maximum protection of 80-100% at a dimethyl sulfoxide concentration of 1.28 M at 77 K. At 77 K, H atoms, ion holes, and electrons can migrate through frozen cells but OH radicals cannot diffuse. Thus the protective effects of dimethyl sulfoxide against cell killing, chromosomal aberrations, and DNA single-strand breaks at 77 K may be due to the scavenging of H atoms or other ions, rather than OH radicals.     

Free radicals photosensitized by near-UV radiation in aqueous adenine solutions in the presence of inorganic phosphate at 77 K

It was shown that irradiation of aqueous adenine solutions in the presence of inorganic phosphate by near UV at 77 K results in the production of OH* free radicals catalyzed by phosphate and photosensitized formation of phosphate radicals and atoms H*.     

Measurements of alkali-labile DNA damage and protein-DNA crosslinks after 2450 MHz microwave and low-dose gamma irradiation in vitro

In vitro experiments were performed to determine whether 2450 MHz microwave radiation induces alkali-labile DNA damage and/or DNA-protein or DNA-DNA crosslinks in C3H 10T(1/2) cells. After a 2-h exposure to either 2450 MHz continuous-wave (CW) microwaves at an SAR of 1.9 W/kg or 1 mM cisplatinum (CDDP, a positive control for DNA crosslinks), C3H 10T(1/2) cells were irradiated with 4 Gy of gamma rays ((137)Cs). Immediately after gamma irradiation, the single-cell gel electrophoresis assay was performed to detect DNA damage. For each exposure condition, one set of samples was treated with proteinase K (1 mg/ml) to remove any possible DNA-protein crosslinks. To measure DNA-protein crosslinks independent of DNA-DNA crosslinks, we quantified the proteins that were recovered with DNA after microwave exposure, using CDDP and gamma irradiation, positive controls for DNA-protein crosslinks. Ionizing radiation (4 Gy) induced significant DNA damage. However, no DNA damage could be detected after exposure to 2450 MHz CW microwaves alone. The crosslinking agent CDDP significantly reduced both the comet length and the normalized comet moment in C3H 10T(1/2) cells irradiated with 4 Gy gamma rays. In contrast, 2450 MHz microwaves did not impede the DNA migration induced by gamma rays. When control cells were treated with proteinase K, both parameters increased in the absence of any DNA damage. However, no additional effect of proteinase K was seen in samples exposed to 2450 MHz microwaves or in samples treated with the combination of microwaves and radiation. On the other hand, proteinase K treatment was ineffective in restoring any migration of the DNA in cells pretreated with CDDP and irradiated with gamma rays. When DNA-protein crosslinks were specifically measured, we found no evidence for the induction of DNA-protein crosslinks or changes in amount of the protein associated with DNA by 2450 MHz CW microwave exposure. Thus 2-h exposures to 1.9 W/ kg of 2450 MHz CW microwaves did not induce measurable alkali-labile DNA damage or DNA-DNA or DNA-protein crosslinks.     

The formation and reaction of methyl radicals produced by direct photolysis of aqueous solutions of N-acetyl substituted aliphatic amino acids at 77K.

Photolysis of amino acids, peptides and their derivatives leads to the formation of free radicals in these substances. The electron-spin-resonance spectra obtained directly after irradiation at 77 K are not very well resolved. They are recognizable as the superposition of the spectra of different types of photoproduced radicals. CH3 radicals are formed by U.V. irradiation if methyl groups are present in the molecule. These radicals are easily detectable because of their four line spectrum. In this paper the formation of methyl radicals and their reaction with undamaged molecules of N-acetyl-substituted amino acids in investigated. The number of CH3 radicals present after a 30 min U.V. irradiation is higher if preceding U.V. irradiations and heat treatments are performed. The overall concentration of radicals is reduced only partially during this heat-treatment, while the CH3 radicals decay completely. Other experiments show that the rate of and the yield of CH3 radicals by U.V. irradiation increase with the dose of a preceding gamma-irradiation. The results suggest that there are substances present which are responsible for the higher production rate of methyl radicals after a preceding treatment. It is assumed that radicals are precursors of the fast-formed CH3 radicals     

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.     

Mammalian cells are not killed by DNA single-strand breaks caused by hydroxyl radicals from hydrogen peroxide

Cell killing by ionizing radiation has been shown to be caused by hydroxyl free radicals formed by water radiolysis. We have previously suggested that the killing is not caused by individual OH free radicals but by the interaction of volumes of high radical density with DNA to cause locally multiply damaged sites (LMDS) (J. F. Ward, Radiat. Res. 86, 185-195, 1985). Here we test this hypothesis using hydrogen peroxide as an alternate source of OH radicals. The route to OH production from H2O2 is expected to cause singly damaged sites rather than LMDS. Chinese hamster V79-171 cells were treated with H2O2 at varying concentrations for varying times at 0 degree C. DNA damage produced intracellularly was measured by alkaline elution and quantitated in terms of Gray-equivalent damage by comparing the rate of its elution with that of DNA from gamma-irradiated cells. The yield of DNA damage produced increases with increasing concentration of H2O2 and with time of exposure. H2O2 is efficient in producing single-strand breaks; treatment with 50 microM for 30 min produces damage equivalent to that formed by 10 Gy of gamma irradiation. In the presence of a hydroxyl radical scavenger, dimethyl sulfoxide (DMSO), the yield of damage decreases with increasing DMSO concentration consistent with the scavenging of hydroxyl radicals traveling an average of 15 A prior to reacting with the DNA. In contrast to DNA damage production, cell killing by H2O2 treatment at 0 degree C is inefficient. Concentrations of 5 X 10(-2) M H2O2 for 10 min are required to produce significant cell killing; the DNA damage yield from this treatment can be calculated to be equivalent to 6000 Gy of gamma irradiation. The conclusion drawn is that individual DNA damage sites are ineffectual in killing cells. Mechanisms are suggested for killing at 0 degree C at high concentrations and for the efficient cell killing by H2O2 at 37 degrees C at much lower concentrations.     

EPR studies of 5-bromouracil crystal after irradiation with X rays in the bromine K-edge region

Radicals induced in a single crystal of 5-bromouracil (BrUra) by synchrotron soft X rays in the bromine K-edge region (13.461-13.482 keV) were investigated using the X-band EPR method. The crystal was irradiated at three peak energies of the absorption spectrum at room temperature or at 80 K. A hydrogen abstraction radical derived from N1 of the pyrimidine ring was commonly observed for all of the energies used, though with some variation in quantity. Similar characteristics were also observed in the EPR signal for the off-K-edge low-energy (13.42 keV) and (60)Co gamma rays used for comparison. When irradiated at 80 K, a much larger exposure (roughly 10 times) of soft X rays was needed to obtain the same signal intensity as that observed at room temperature. EPR signals were not detectable with gamma irradiation at liquid nitrogen temperature.     

Investigations on radical formation and inactivation of suspended trypsin afterγ-irradiation

Summary Crystalline trypsin was irradiated in oxygen-free suspension media of methanol, ethanol and n-heptane with⁶⁰Co--rays at 77 K or 273 K. Measurements with ESR and activity determinations revealed no influence of ethanol and n-heptane on the formation of free radicals and inactivation of trypsin. Especially, the results are independent on the polarity of the suspension media and correspond to an irradiation of trypsin in vacuum. On the other hand, methanol leads to a decay of radiation induced radicals and to an increased inactivation. The results are discussed in comparison to analogous experiments carried out with ultra-violet light.     

The decomposition of the EPR spectra of multicomponent systems irradiated at 77 K into paramagnetic center signals of different natures]

Main principles of the way to decompose an EPR spectrum of a multicomponent system, irradiated at 77 K, into separate radiation-induced paramagnetic centre signals are given. The decomposition is possible due to the computer assistant spectra processing, and is based on different properties of different paramagnetic centres, namely, on different thermostability of the centres, on different rate of relaxation, and on different photosensitivity. Concrete examples of the EPR spectrum decomposition into different free radical signals are given for cases of murine liver and spleen irradiated at 77 K. Radiochemical yields of different free radicals, induced by gamma radiation at 77 K in whole biological tissues, were defined. The data on nature and properties of the paramagnetic centres induced by radiation in biological tissues are shortly reviewed.     

Influence of chromatin structure on induction of double-strand breaks in mammalian cells irradiated with DNA-incorporated 125I

In this study the induction of double-strand breaks (DSBs) was investigated in Chinese hamster V79-379A cells irradiated with the Auger-electron emitter (125)I incorporated into DNA. The role of chromatin organization was studied by pulse-labeling synchronized cells with (125)IdU before decay accumulation in early or late S phase. Pulsed-field gel electrophoresis and fragment-size analysis were used to quantify the distribution of DNA fragments in irradiated intact cells and naked DNA as well as in DNA from asynchronously labeled cultures in a different scavenging environment. The results show that in intact cells, after accumulation of decays at -70 degrees C in the presence of 10% DMSO, almost four times more DSBs were induced in late S phase compared with early S phase and the fragment distribution was clearly non-random with an excess of fragments <0.2 Mbp. The DSB yield was 0.6 DSB/cell and decay for cells irradiated in early S phase and 2.3 DSBs/cell and decay for cells irradiated in late S phase. When similar experiments were performed on naked genomic DNA or intact cells irradiated with gamma rays, the difference in yield was not as prominent. These data imply a role of chromatin organization in the induction of DSBs by DNA-incorporated (125)I. In summary, the results presented here suggest that the yield of DSBs as well as the fragment distribution induced by (125)IdU decay may vary significantly depending on the chromatin organization during S phase and the labeling procedure used.     

Comparison of stochastic and deterministic methods for modeling spur kinetics

Stochastic and deterministic kinetic methods have been used to model the temporal evolution of spatially nonhomogeneous clusters of reactants resulting from the dissociation of one to six water molecules into either H3O+, OH, and e-aq, or H atoms and OH radicals. When the ionic fragmentation initially producing H3O+, OH, and e-aq is considered, the stochastic and deterministic methods predict similar time dependences for the decay of the reactive species; however, the two methods suggest very different product yields. For a two-dissociation spur, the deterministic treatment overestimates both the H2 and the H2O2 yields by about 70%. The error decreases to less than 15% for a spur with six water dissociations. For a distribution of spurs representing a high-energy electron track, the differences in the predicted yields of reactants are less than 6% at 0.1 microseconds, but the stochastic and deterministic predictions for the yields of H2 and H2O2 differ by about 50%. The kinetics of spurs produced by the fragmentation of water to H atom and OH radical shows discrepancies in both the reactant and the product yields. The size of the discrepancy decreases as the number of H/OH pairs increases, and the predictions of the two techniques are almost the same for clusters of six water dissociations.     

OH radicals and oxidizing products in the gamma radiolysis of water

The production of OH radicals in the gamma radiolysis of water has been examined with radical scavenger techniques employing formic acid. OH radical yields were found to vary from 2.4 radicals/100 eV at the low scavenger concentration limit to 4.2 at a formic acid concentration of 3 M. An inverse Laplace transform technique was applied to the scavenger concentration dependence to obtain the temporal dependence of OH radicals in pure water. It was found that the relative decrease in OH radical yields from 200 ps to 3 ns was virtually the same for the transform of the scavenger data and the directly measured time-resolved results. The absolute yields for the time-resolved experiments are about 10% higher than expected from the present results with scavengers. The agreement can be considered to be good, and reasons for the observed difference are given. Approximately 40% of the OH radicals produced lead to the formation of hydrogen peroxide, which is the only other major oxidizing species in the gamma radiolysis of water. The net water decomposition for gamma rays was found to vary from an initial value of 5.6 +/- 0.3 molecules/100 eV to 3.8 +/- 0.2 molecules/100 eV at 1 micros.     

Relative biological effectiveness of the alpha-particle emitter (211)At for double-strand break induction in human fibroblasts

The purpose of this study was to quantify and to determine the distribution of DNA double-strand breaks (DSBs) in human cells irradiated in vitro and to evaluate the relative biological effectiveness (RBE) of the alpha-particle emitter (211)At for DSB induction. The influence of the irradiation temperature on the induction of DSBs was also investigated. Human fibroblasts were irradiated as intact cells with alpha particles from (211)At, (60)Co gamma rays and X rays. The numbers and distributions of DSBs were determined by pulsed-field gel electrophoresis with fragment analysis for separation of DNA fragments in sizes 10 kbp-5.7 Mbp. A non-random distribution was found for DSB induction after irradiation with alpha particles from (211)At, while irradiation with low-LET radiation led to more random distributions. The RBEs for DSB induction were 2.1 and 3.1 for (60)Co gamma rays and X rays as the reference radiation, respectively. In the experiments studying temperature effects, nuclear monolayers were irradiated with (211)At alpha particles or (60)Co gamma rays at 2 degrees C or 37 degrees C and intact cells were irradiated with (211)At alpha particles at the same temperatures. The dose-modifying factor (DMF(temp)) for irradiation of nuclear monolayers at 37 degrees C compared with 2 degrees C was 1.7 for (211)At alpha particles and 1.6 for (60)Co gamma rays. No temperature effect was observed for intact cells irradiated with (211)At. In conclusion, irradiation with alpha particles from (211)At induced two to three times more DSB than gamma rays and X rays.     

Metal-independent production of hydroxyl radicals by halogenated quinones and hydrogen peroxide: an ESR spin trapping study

The metal-independent production of hydroxyl radicals (*OH) from H(2)O(2) and tetrachloro-1,4-benzoquinone (TCBQ), a carcinogenic metabolite of the widely used wood-preservative pentachlorophenol, was studied by electron spin resonance methods. When incubated with the spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO), TCBQ and H(2)O(2) produced the DMPO/*OH adduct. The formation of DMPO/*OH was markedly inhibited by the *OH scavenging agents dimethyl sulfoxide (DMSO), ethanol, formate, and azide, with the concomitant formation of the characteristic DMPO spin trapping adducts with *CH(3), *CH(CH(3))OH, *COO(-), and *N(3), respectively. The formation of DMPO/*OH and DMPO/*CH(3) from TCBQ and H(2)O(2) in the absence and presence, respectively, of DMSO was inhibited by the trihydroxamate compound desferrioxamine, accompanied by the formation of the desferrioxamine-nitroxide radical. In contrast, DMPO/*OH and DMPO/*CH(3) formation from TCBQ and H(2)O(2) was not affected by the nonhydroxamate iron chelators bathophenanthroline disulfonate, ferrozine, and ferene, as well as the copper-specific chelator bathocuproine disulfonate. A comparative study with ferrous iron and H(2)O(2), the classic Fenton system, strongly supports our conclusion that *OH is produced by TCBQ and H(2)O(2) through a metal-independent mechanism. Metal-independent production of *OH from H(2)O(2) was also observed with several other halogenated quinones.     

Theoretical investigation of the formation of a new series of antioxidant depsides from the radiolysis of flavonoid compounds

This paper deals with the formation of a series of antioxidant depsides obtained from flavonoid solutions irradiated with gamma rays. These reactions take place in radiolyzed alcohol solutions, a medium that is very rich in many different highly reactive species and that hosts specific reactions. We focus on the first step of those reactions, i.e., reactivity of the solute (flavonoid) with the alkoxy radicals CH(3)O(*) and CH(3)CH(2)O(*) formed in methanol and ethanol, respectively, and their carbon-centered isomers: the 1-hydroxy-methyl ((*)CH(2)OH) and the 1-hydroxy-ethyl (CH(3)(*)CHOH) radicals. Among the different flavonoid groups of molecules, only flavonols are transformed. To establish the structure-reactivity relationship that explains why the radiolytic transformation occurs only for those compounds, the process is rationalized theoretically, with Density Functional Theory calculations, taking into account the solvent effects by a Polarizable Continuum Model and a microhydrated environment (one or two water molecules surrounding the active center). The first redox reaction, occurring between the flavonol and the reactive species formed upon irradiation of the solvent, is studied in terms of (1) the O-H bond dissociation enthalpy of each OH group of the flavonoids and (2) electron abstraction from the molecule. We conclude that the reaction, initiated preferentially by the alkoxy radicals, first occurs at the 3-OH group of the flavonol. It is then followed by the formation of a peroxyl radical (after molecular oxygen or superoxide addition). The different cascades of reactions, which lead to the formation of depsides via C-ring opening, are discussed on the basis of the corresponding calculated energetic schemes.     

Thrombomodulin as a marker of radiation-induced endothelial cell injury.

Cultured confluent human umbilical vein endothelial cells were irradiated in vitro with 60Co gamma rays at doses from 0 to 50 Gy. After irradiation thrombomodulin was measured at different times over 6 days in the supernatants of endothelial cell culture medium, on the surface of the cells, and within the cells. At 24 h after irradiation, an increase in the release of thrombomodulin from irradiated endothelial cells and an increase in the number of molecules and the activity of thrombomodulin on the surface of the cells were observed; these reactions were dependent on radiation dose. The capacity of the cells to produce and release thrombomodulin was decreased from 2 to 6 days after exposure to 60Co gamma rays. Our data indicate that radiation can injure endothelial cells, and that thrombomodulin may be used as a marker of radiation-induced injury in endothelial cells. The interrelationship between the dysfunction of irradiated endothelial cells and the pathological mechanisms of acute radiation disease is also discussed.     

Repair of damage in double-stranded phi X174 (RF) DNA due to radiation-induced water radicals

Experiments in which the yields of radiation-induced OH and H radicals were varied, showed that both types of water radicals inactivate phi X174 RF DNA to about the same extent as measured by transfection of the (irradiated) DNA to E. coli wild-type spheroplasts. On the other hand, using spheroplasts prepared from E. coli strains, deficient in one of the proteins involved in excision DNA repair (uvrA- or uvrC-) or in post-replication repair (recA-), clear differences between damage originating from OH or H radical attack were found. Part of the radiation damage due to H radicals appeared to be repairable by an uvrA-gene-dependent repair mechanism, whereas this repair pathway does not play an important role in the case of OH radical damage. The reverse applies to uvrC-gene-dependent repair, which only affects OH radical damage (obtained under anoxic conditions), but has no influence on damage due to H radicals. Irradiation of double-stranded phi X174 (RF) DNA in the presence of oxygen however, yields damage--due to OH radicals only--which appeared not to be sensitive to either uvrC- or uvrA-gene-dependent repair. Furthermore, post-replication repair (recA) has only very little effect on the amount of inactivation by H or OH radicals, when irradiation is carried out under anoxic conditions. We did not find significant inactivation due to hydrated electrons, whether the biological activity was determined by use of wild-type spheroplasts or of strains deficient in excision or post-replication repair proteins.     

Radioprotection by DMSO of mammalian cells exposed to X-rays and to heavy charged-particle beams

Populations of G1-phase Chinese hamster cells in stirred suspensions containing various concentrations of DMSO were irradiated with 250 kV X-rays or various heavy charged-particle beams. Chemical radioprotection of cell inactivation was observed for all LET values studied. When cell survival data were resolved into linear and quadratic components, the extent and concentration dependence of DMSO protection were found to be different for the two mechanisms. The chemical kinetics of radioprotection for single-events were similar for LET values up to those which gave the maximum RBE. DMSO protected to a lesser extent against energetic argon ions at an median LET of approximately 220 keV/micron. These data could indicate the contribution of indirect action by hydroxyl radicals and hydrogen atoms to cell inactivation by single-hit and double-hit mechanisms for various radiation qualities. The decrease in RBE observed at very high LET may result, in part, from reduced yields of water radicals at 10(-9)-10(-8) s resulting from radical recombination mechanisms within the charged particle tracks.     

Dependence of the nucleoside effect on linear energy transfer.

L929 cells were irradiated by cyclotron-produced neutrons and by 14.8 MeV monoenergetic neutrons. For comparison cells were also irradiated by 60Co gamma rays. Following irradiation cells were treated by an equimolar solution of deoxyribonucleosides, and the effect on cell survival measured. Results show that nucleoside treatment was efficient after low-LET irradiation: gamma ray survival curves were altered by deoxyribonucleosides in terms of significantly increased extrapolation numbers only, but without Do change. Cells irradiated by neutrons from either of the two sources did not respond to nucleoside treatment, and consequently their survival curves remained unaltered. These results show that the nucleoside effect does occur after low-LET irradiation, but apparently not following high-LET irradiation. Since deoxyribonucleosides as well as other cell breakdown products are released in irradiated and necrotic tumours due to massive cell destruction, such a nucleoside effect could possibly enhance the cell survival and thus effect the result of radiotherapy. Absence of the nucleoside effect in case of high-LET irradiation may therefore be an additional potential gain from neutrons in radiotherapy.     

Influence of hyperthermia on gamma-ray-induced mutation in V79 cells

Asynchronously growing V79 cells were assayed for mutation induction following exposure to hyperthermia either immediately before or after being irradiated with 60Co gamma rays. Hyperthermia exposures consisted of either 43.5 degrees C for 30 min or 45 degrees C for 10 min. Each of these heat treatments resulted in a survival level of 42%. For all sequences of combined treatment with hyperthermia and radiation, cell killing by gamma rays was enhanced. Mutation induction by gamma rays was enhanced when heat preceded gamma irradiation, but no increase was observed when heat was given after gamma exposures. Treatment at 45 degrees C for 10 min gave a higher yield in mutants at all gamma doses studied compared to treatment at 43.5 degrees C for 30 min. When heat-treated cells were incubated for different periods before being exposed to gamma rays, thermal enhancement of radiation killing was lost after 24 h. In contrast, only 5-6 h incubation was needed for loss of mutation induction enhancement.