The role of the DMPO-hydrated electron spin adduct in DMPO-*OH spin trapping

Time-resolved in situ radiolysis ESR (electron spin resonance, equivalently EPR, electron paramagnetic resonance) studies have shown that the scavenging of radiolytically produced hydroxyl radical in nitrous oxide-saturated aqueous solutions containing 2 mM DMPO is essentially quantitative (94% of the theoretical yield) at 100 micros after the electron pulse [1]. This result appeared to conflict with earlier results using continuous cobalt-60 gamma radiolysis and hydrogen peroxide photolysis, where factors of 35 and 33% were obtained, respectively [2,3]. To investigate this discrepancy, nitrogen-saturated aqueous solutions containing 15 mM DMPO were cobalt-60 gamma irradiated (dose rate = 223 Gy/min) for periods of 0.25-6 min, and ESR absorption spectra were observed approximately 30 s after irradiation. A rapid, pseudo-first-order termination reaction of the protonated DMPO-hydrated electron adduct (DMPO-H) with DMPO-OH was observed for the first time. The rate constant for the reaction of DMPO-H with DMPO-OH is 2.44 x 10(2) (+/- 2.2 x 10(1)) M(-1) s(-1). In low-dose radiolysis experiments, this reaction lowers the observed yield of DMPO-OH to 44% of the radiation-chemical OH radical yield (G = 2.8), in good agreement with the earlier results [2,3]. In the absence of the DMPO-H radical, the DMPO-OH exhibits second-order radical termination kinetics, 2k(T) = 22 (+/- 2) M(-1) s(-1) at initial DMPO-OH concentrations > or = 13 microM, with first-order termination kinetics observed at lower concentrations, in agreement with earlier literature reports [4].     

Direct evidence for in vivo hydroxyl radical generation in blood of mice after acute chromium (VI) intake

Although it is assumed from in vitro experiments that the hydroxyl radical (*OH) may be responsible for chromium(VI) toxicity/carcinogenicity, no electron spin resonance (ESR) evidence for the generation of *OH in vivo has been reported. In this study, we have employed an ESR spin-trapping technique with 5,5-dimethylpyrroline-N-oxide (DMPO), a selective *OH trap, to detect *OH in blood. The ESR spectrum of spin adduct observed in the blood of mice given 4.8 mmol Cr(VI)/kg body weight exhibited the 1:2:2:1 intensity pattern of a quartet with a hyperfine coupling constant A(N) = A(H) = 14.81 G and g-value = 2.0067. The concentration of the spin adduct detected in the blood was 7.37 microM. The adduct production was inhibited by the addition of specific *OH scavengers such as sodium benzoate and methional to the blood. The results indicate that the spin adduct is nitroxide produced by the reaction of *OH with DMPO. This is the first report of ESR evidence for the in vivo generation of *OH in mammals by Cr(VI).     

High-LET ion radiolysis of water: visualization of the formation and evolution of ion tracks and relevance to the radiation-induced bystander effect

Ionizing radiation-induced bystander effects, commonly observed in cell populations exposed to high-linear energy transfer (LET) radiations, are initiated by damage to a cellular molecule which then gives rise to a toxic signal exported to neighboring cells not directly hit by radiation. A major goal in studies of this phenomenon is the identification of this initial radiation-induced lesion. Liquid water being the main constituent of biological matter, reactive species produced by water radiolysis in the cellular environment are likely to be major contributors to the induction of this lesion. In this context, the radiation track structure is of crucial importance in specifying the precise location and identity of all the radiolytic species and their subsequent signaling or damaging effects. We report here Monte Carlo track structure simulations of the radiolysis of liquid water by four different impacting ions 1H+, 4He2+, 12C6+ and 20Ne10+, with the same LET ( approximately 70 keV/ microm). The initial radial distribution profiles of the various water decomposition products (eaq(-), *OH, H*, H2 and H2O2) for the different ions considered are presented and discussed briefly in the context of track structure theory. As an example, the formation and temporal evolution of simulated 24 MeV 4He2+ ion tracks (LET approximately 26 keV/microm) are reported for each radiolytic species from 1 ps to 10 micros. The calculations reveal that the ion track structure is completely lost by approximately 1 micros.     

Mutagenic effects of carbon ions near the range end in plants

To gain insight into the mutagenic effects of accelerated heavy ions in plants, the mutagenic effects of carbon ions near the range end (mean linear energy transfer (LET): 425keV/μm) were compared with the effects of carbon ions penetrating the seeds (mean LET: 113keV/μm). Mutational analysis by plasmid rescue of Escherichia coli rpsL from irradiated Arabidopsis plants showed a 2.7-fold increase in mutant frequency for 113keV/μm carbon ions, whereas no enhancement of mutant frequency was observed for carbon ions near the range end. This suggested that carbon ions near the range end induced mutations that were not recovered by plasmid rescue. An Arabidopsis DNA ligase IV mutant, deficient in non-homologous end-joining repair, showed hyper-sensitivity to both types of carbon-ion irradiation. The difference in radiation sensitivity between the wild type and the repair-deficient mutant was greatly diminished for carbon ions near the range end, suggesting that these ions induce irreparable DNA damage. Mutational analysis of the Arabidopsis GL1 locus showed that while the frequency of generation of glabrous mutant sectors was not different between the two types of carbon-ion irradiation, large deletions (>～30kb) were six times more frequently induced by carbon ions near the range end. When 352keV/μm neon ions were used, these showed a 6.4 times increase in the frequency of induced large deletions compared with the 113keV/μm carbon ions. We suggest that the proportion of large deletions increases with LET in plants, as has been reported for mammalian cells. The nature of mutations induced in plants by carbon ions near the range end is discussed in relation to mutation detection by plasmid rescue and transmissibility to progeny.     

Contribution of indirect action to radiation-induced mammalian cell inactivation: dependence on photon energy and heavy-ion LET

The contribution of indirect action mediated by OH radicals to cell inactivation by ionizing radiations was evaluated for photons over the energy range from 12.4 keV to 1.25 MeV and for heavy ions over the linear energy transfer (LET) range from 20 keV/microm to 440 keV/microm by applying competition kinetics analysis using the OH radical scavenger DMSO. The maximum level of protection provided by DMSO (the protectable fraction) decreased with decreasing photon energy down to 63% at 12.4 keV. For heavy ions, a protectable fraction of 65% was found for an LET of around 200 keV/microm; above that LET, the value stayed the same. The reaction rate of OH radicals with intracellular molecules responsible for cell inactivation was nearly constant for photon inactivation, while for the heavy ions, the rate increased with increasing LET, suggesting a reaction with the densely produced OH radicals by high-LET ions. Using the protectable fraction, the cell killing was separated into two components, one due to indirect action and the other due to direct action. The inactivation efficiency for indirect action was greater than that for direct action over the photon energy range and the ion LET range tested. A significant contribution of direct action was also found for the increased RBE in the low photon energy region.     

Monte Carlo calculation of the primary radical and molecular yields of liquid water radiolysis in the linear energy transfer range 0.3-6.5 keV/micrometer: application to 137Cs gamma rays

Monte Carlo simulations of the radiolysis of neutral liquid water and 0.4 M H(2)SO(4) aqueous solutions at ambient temperature are used to calculate the variations of the primary radical and molecular yields (at 10(-6)s) as a function of linear energy transfer (LET) in the range approximately 0.3 to 6.5 keV/micrometer. The early energy deposition is approximated by considering short (approximately 20-100 micrometer) high-energy (approximately 300-6.6 MeV) proton track segments, over which the LET remains essentially constant. The subsequent nonhomogeneous chemical evolution of the reactive species formed in these tracks is simulated by using the independent reaction times approximation, which has previously been used successfully to model the radiolysis of water under various conditions. The results obtained are in good general agreement with available experimental data over the whole LET range studied. After normalization of our computed yields relative to the standard radical and molecular yields for (60)Co gamma radiation (average LET approximately 0.3 keV/micrometer), we obtain empirical relationships of the primary radiolytic yields as a function of LET over the LET range studied. Such relationships are of practical interest since they allow us to predict a priori values of the radical and molecular yields for any radiation from the knowledge of the average LET of this radiation only. As an application, we determine the corresponding yields for the case of (137)Cs gamma radiation. For this purpose, we use the value of approximately 0.91 keV/micrometer for the average LET of (137)Cs gamma rays, chosen so that our calculated yield G(Fe(3+)) for ferrous-ion oxidation in air-saturated 0.4 M sulfuric acid reproduces the value of 15.3 molecules/100 eV for this radiation recommended by the International Commission on Radiation Units and Measurements. The uncertainty range on those primary radical and molecular yields are also determined knowing the experimental error (approximately 2%) for the measured G(Fe(3+)) value. The following values (expressed in molecules/100 eV) are obtained: (1) for neutral water: G(e(-)(aq)) = 2.50 +/- 0.16, G(H(.)) = 0.621 +/- 0.019, G(H(2)) = 0.474 +/- 0.025, G((.)OH) = 2.67 +/- 0.14, G(H(2)O(2)) = 0.713 +/- 0.031, and G(-H(2)O) = 4.08 +/- 0.22; and (2) for 0.4 M H(2)SO(4) aqueous solutions: G(H(.)) = 3.61 +/- 0.09, G(H(2)) = 0.420 +/- 0.019, G((.)OH) = 2.78 +/- 0.12, G(H(2)O(2)) = 0.839 +/- 0.037, and G(-H(2)O) = 4.46 +/- 0.16. These computed values are found to differ from the standard yields for (60)Co gamma rays by up to approximately 6%.     

Simulation of DNA damage after proton irradiation

The biophysical radiation track simulation model PARTRAC was improved by implementing new interaction cross sections for protons in water. Computer-simulated tracks of energy deposition events from protons and their secondary electrons were superimposed on a higher-order DNA target model describing the spatial coordinates of the whole genome inside a human cell. Induction of DNA double-strand breaks was simulated for proton irradiation with LET values between 1.6 and 70 keV/microm and various reference radiation qualities. The yield of DSBs after proton irradiation was found to rise continuously with increasing LET up to about 20 DSBs per Gbp and Gy, corresponding to an RBE up to 2.2. About half of this increase resulted from a higher yield of DSB clusters associated with small fragments below 10 kbp. Exclusion of experimentally unresolved multiple DSBs reduced the maximum DSB yield by 30% and shifted it to an LET of about 40 keV/microm. Simulated fragment size distributions deviated significantly from random breakage distributions over the whole size range after irradiation with protons with an LET above 10 keV/microm. Determination of DSB yields using equations derived for random breakage resulted in an underestimation by up to 20%. The inclusion of background fragments had only a minor influence on the distribution of the DNA fragments induced by radiation. Despite limited numerical agreement, the simulations reproduced the trends in proton-induced DNA DSBs and fragment induction found in recent experiments.     

Formation of .OH and .H in aqueous solutions by ultrasound using clinical equipment

Hydroxyl radicals and hydrogen atoms were produced in argon-saturated aqueous solutions exposed to ultrasound using clinical dental equipment. .OH and .H radicals were detected and identified by ESR and were spin trapped with 5,5-dimethyl-1-delta-pyrroline-N-oxide (DMPO) and alpha-4-pyridyl-1-oxide-N-tert-butylnitrone (POBN). The observed ESR spectra were compared with those obtained from sonolysis of argon-saturated water in an ultrasonic bath, from gamma radiolysis of air-saturated water, and from uv photolysis of aqueous hydrogen peroxide solutions.     

Comparison of inhibitory effects of oxygen radicals and calf serum protein on surfactant activity

The effects of the reactive oxygen species (ROS) superoxide anion (O2*-) and hydroxyl radical (*OH) on the surface tension lowering properties of bovine lipid extract surfactant (BLES) were compared to the effects of calf serum protein (CSP) in a captive bubble surfactometer (CBS). O2*- was generated from xanthine/xanthine oxidase (X/XO), and *OH was generated by the Fenton reaction. ROS were demonstrated by electron spin resonance (ESR) using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap. Lipid peroxidation was measured using the thiobarbituric acid method. *OH had broad inhibitory effects on surface tension parameters, including adsorption, minimum surface tension, percentage film area change and film compressibility. O2*- showed inhibitory effects on adsorption, film area change and film compressibility but had no significant effect on minimum surface tension. Both O2*- and *OH treatment were associated with a large 'squeezeout' plateau around 20-25 mN/m in the surface tension-area relation, indicating poor film organization during the compression phase. At the concentrations used, ROS were associated with lipid peroxidation of BLES, which also demonstrated radical scavenging properties. Calf serum protein produced inhibitory effects on adsorption, minimum surface tension and percentage film area change that were quantitatively similar to those produced by *OH. The effects on film compression were significantly greater and qualitatively different from those seen with either O2*- or *OH. We conclude that the inhibition of BLES surface activity by ROS and inhibitory proteins can be distinguished in the captive bubble surfactometer and, particularly, by changes in the film compressibility modulus.     

The quality of DNA double-strand breaks: A Monte Carlo simulation of the end-structure of strand breaks produced by protons and alpha particles

The quality of DNA damage induced by protons and -particles of various linear energy transfer (LET) was studied. The aim was to single out specific lesions in the DNA molecule that might lead to biological endpoints such as inactivation. A DNA model coupled with a track structure code (MOCA-15) were used to simulate the lesions induced on the two helixes. Four categories of DNA breaks were considered: single-strand breaks (ssb), bluntended double-strand breaks (dsb, with no or few overlapping bases), sticky-ended double-strand breaks (with cohesive free ends of many bases), and deletions (complex lesions which involve at least two dsb within a small number of base pairs). Calculations were carried out assuming various sets of parameters characterizing the production of these different DNA breaks. No large variations in the yields of ssb and blunt- or sticky-ended dsb were found in the LET range between 10 and 200 keV/µm. On the other hand, the yield of deletions increases up to about 100 keV/µm and seems to reach a plateau at higher LET values. In the LET interval from 30 to 60 keV/µm, protons proved to be more efficient than -particles in inducing deletions. The induction of these complex lesions is thus dependent not simply on LET but also on the characteristics of the track structure. Comparison with RBE values for cell killing shows that this special class of dsb might play an important role in radiation-induced cell inactivation.     

Detection of heavy-ion-induced DNA double-strand breaks using static-field gel electrophoresis

Radiation-induced DNA double-strand breaks (DSBs) were measured in Chinese hamster ovary cells (CHO-K1) using an experimental protocol involving static-field gel electrophoresis following exposure to various accelerated ions. Dose-effect curves were set up, and relative biological efficiencies (RBEs) for DSB induction were determined for different radiation qualities. RBEs around 1 were obtained for low energy deuterons (6–7 keV/µm), while for high energy oxygen ions (20keV/µm) an RBE value slightly greater than 1 was determined. Low energetic oxygen ions (LET=250 keV/µm) were found to show RBEs substantially below unity, and for higher LET particles (31 y-250 keVµm) RBEs for DSB induction were generally found to be smaller than 1. The data presented here are in line with the generally accepted view that not induced DSBs, but rather misrepaired or unrepaired DNA lesions are related to cellular inactivation.     

Effect of multiple ionization on the yield of H2O2 produced in the radiolysis of aqueous 0.4 M H2SO4 solutions by high-LET 12C6+ and 20Ne9+ ions

Monte Carlo track structure simulations were performed to investigate the effect of multiple ionization of water on the primary (or "escape") (at approximately 10(-6) s) yield of hydrogen peroxide (G(H2O2)) produced in the radiolysis of deaerated 0.4 M H2SO4 solutions by 12C6+ and 20Ne9+ ions at high linear energy transfer (LET) up to approximately 900 keV/microm. It was found that, upon incorporating the mechanisms of double, triple and quadruple ionizations of water in the calculations, a quantitative agreement between theory and experiment can be obtained. The curve for G(H2O2) as a function of LET reaches a well-defined maximum of approximately 1.4 molecules/100 eV at approximately 180-200 keV/microm, in very good accord with the available experimental data. Our results also show that, for the highest LET values considered in this study, the H2O2 escape yields obtained in 0.4 M sulfuric acid solutions are about 45% greater in magnitude than those found in neutral water. Contrary to a recent assumption suggesting that the limiting value of G(H2O2) at infinite LET should be approximately 1 molecule/100 eV, somewhat similar for neutral and acidic water, our simulations show a clear decrease in the primary H2O2 yields with increasing LET at high LET, indicating that the question of the limiting value of G(H2O2) at very high LET for both neutral and acidic liquid water is still open.     

Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to low doses of heavy-ion radiation

Adaptive response (AR) and bystander effect are two important phenomena involved in biological responses to low doses of ionizing radiation (IR). Furthermore, there is a strong interest in better understanding the biological effects of high-LET radiation. We previously demonstrated the ability of low doses of X-rays to induce an AR to challenging heavy-ion radiation [8]. In this study, we assessed in vitro the ability of priming low doses (0.01Gy) of heavy-ion radiation to induce a similar AR to a subsequent challenging dose (1-4Gy) of high-LET IR (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm) in TK6, AHH-1 and NH32 cells. Our results showed that low doses of high-LET radiation can induce an AR characterized by lower mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and faster DNA repair kinetics, in cells expressing p53.     

Cytogenetic effects of densely ionising radiation in human lymphocytes: impact of cell cycle delays

The classical cytogenetic assay to estimate the dose to which an individual has been exposed relies on the measurement of chromosome aberrations in lymphocytes at the first post-irradiation mitosis 48 h after in vitro stimulation. However, evidence is accumulating that this protocol results in an underestimation of the cytogenetic effects of high LET radiation due to a selective delay of damaged cells. To address this issue, human lymphocytes were irradiated with C-ions (25-mm extended Bragg peak, LET: 60-85 keV/ micro m) and aberrations were measured in cells reaching the first mitosis after 48, 60, 72 and 84 h and in G2-phase cells collected after 48 h by calyculin A induced premature chromosome condensation (PCC). The results were compared with recently published data on the effects of X-rays and 200 MeV/u Fe-ions (LET: 440 keV/ micro m) on lymphocytes of the same donor (Ritter et al., 2002a). The experiments show clearly that the aberration yield rises in first-generation metaphase (M1) with culture time and that this effect increases with LET. Obviously, severely damaged cells suffer a prolonged arrest in G2. The mitotic delay has a profound effect on the RBE: RBE values estimated from the PCC data were about two times higher than those obtained by conventional metaphase analysis at 48 h. Altogether, these observations argue against the use of single sampling times to quantify high LET induced chromosomal damage in metaphase cells.     

Qualitatively different induction of germ cell mutations by heavy ions

Summary The problem of the dependence of the biological efficiency of ionizing radiation on the Linear Energy Transfer (LET) is still unsolved. Unexpected reactions of heavy ion irradiated cellular systems such as an increasing Relative Biological Effectiveness (RBE) up to a LET of about 100 keV/µm and then a decrease below 1 oblige to dismiss some conventional interpretations. Several years ago we suggested that, especially by higher ionization density in addition to the DNA, repair systems and (or) membraneous systems could also be injured (dual target theory). Our experiments with heavy ions at the Lawrence Berkeley Laboratory with LET's between 102–970 keV/µm on different types of mutations show a strict distinction between events connected with fusion modalities (repair or misrepair) and those associated with nonfusion. With very high LET misrepair reactions such as translocations disappear, suggesting the direct damage of the repair systems and confirming our previous experiments with peak pions and ions of LET's between 1100–4800 keV/µm.Dedicated to Prof. W. Jacobi on the occasion of his 60th birthday     

Terminal maturation of megakaryocytes and platelet production by hematopoietic stem cells irradiated with heavy-ion beams

Hematopoietic processes, especially megakaryocytopoiesis and thrombopoiesis, are highly sensitive to high-linear energy transfer (LET) radiations such as heavy-ion beams that have greater biological effects than low-LET radiation. This study examined the terminal maturation of megakaryocytes and platelet production derived from hematopoietic stem cells irradiated with heavy-ion beams. CD34(+) cells derived from human placental/umbilical cord blood were exposed to monoenergetic carbon-ion beams (LET = 50 keV/μm) and then cultured in a serum-free medium supplemented with thrombopoietin and interleukin-3. There was no significant difference in megakaryocyte-specific markers between nonirradiated control and irradiated cells. Expression of Tie-2, a receptor that acts in early hematopoiesis, showed a significant 1.31-fold increase after 2 Gy irradiation compared to control cells on day 7. There was a significant increase in Tie-2 mRNA expression. In addition, the expression of other mRNAs, such as PECAM1, SELP and CD44, was also significantly increased in cells irradiated with heavy-ion beams. However, the adherent function of platelets derived from the irradiated cells showed no difference from that in the controls. These results clarify that the functions of megakaryocytopoiesis and thrombopoiesis derived from hematopoietic stem/progenitor cells irradiated with heavy-ion beams are similar to those in the unirradiated cells, although heavy-ion beams affect the expression of genes associated with cellular adhesion.     

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.     

A Kinetic and ESR Investigation of Iron(II) Oxalate Oxidation by Hydrogen Peroxide and Dioxygen as a Source of Hydroxyl Radicals

The reaction of Fe^II oxalate with hydrogen peroxide and dioxygen was studed for oxalate concentrations up to 20 mM and pH 2-5, under which conditions mono- and bis-oxalate comlexes (Fe^II(ox) and Fe^II(ox)₂^2-) and uncomplexed Fe²⁺ must be considered. The reaction of Fe^II oxalate with hydrogen peroxide (Fe²⁺ + H₂O₂ → Fe³⁺ + *OH + OH^-) was monitored in continuous flow by ESR with t-butanol as a radical trap. The reaction is much faster than for uncomplexed Fe²⁺ and a rate constant, k = 1 × 10⁴ M-¹ s^-1 is deduced for Fe^II(ox). The reaction of Fe^II oxalate with dioxygen is strongly pH dependent in a manner which indicates that the reactive species is Fe^II(ox)₂^2-, for which an apparent second order rate constant, k = 3.6 M^-1 s^-1, is deduced. Taken together, these results provide a mechanism for hydroxyl radical production in aqueous systems containing Fe^II complexed by oxalate. Further ESR studies with DMPO as spin trap reveal that reaction of Fe^II oxalate with hydrogen peroxide can also lead to formation of the carboxylate radical anion (CO₂^*-), an assignment confirmed by photolysis of Fe^III oxalate in the presence of DMPO.     

Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to X-rays

The ability of cells to adapt low-dose or low-dose rate radiation is well known. High-LET radiation has unique characteristics, and the data concerning low doses effects and high-LET radiation remain fragmented. In this study, we assessed in vitro the ability of low doses of X-rays to induce an adaptive response (AR) to a subsequent challenging dose of heavy-ion radiation. Lymphoblastoid cells (TK6, AHH-1, NH32) were exposed to priming 0.02-0.1Gy X-rays, followed 6h later by challenging 1Gy heavy-ion radiation (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm). Pre-exposure of p53-competent cells resulted in decreased mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and different H2AX phosphorylation kinetics, as compared to cells exposed to challenging radiation alone. This phenomenon did not seem to be linked with cell cycle effects or radiation-induced apoptosis. Taken together, our results suggested the existence of an AR to mutagenic effects of heavy-ion radiation in lymphoblastoid cells and the involvement of double-strand break repair mechanisms.