Increase of positive supercoiling in a hyperthermophilic archaeon after UV irradiation.

Diverse DNA repair mechanisms are essential to all living organisms. Some of the most widespread repair systems allow recovery of genome integrity in the face of UV radiation. Here, we show that the hyperthermophilic archaeon Thermococcus nautili possesses a remarkable ability to recovery from extreme chromosomal damage. Immediately following UV irradiation, chromosomal DNA of T. nautili is fragmented beyond recognition. However, the extensive UV-induced double-stranded breaks (DSB) are repaired over the course of several hours, allowing restoration of growth. DSBs also disrupted plasmid DNA in this species. Similar to the chromosome, plasmid integrity was restored during an outgrowth period. Intriguingly, the topology of recovered pTN1 plasmids differed from control strain by being more positively supercoiled. As reverse gyrase (RG) is the only enzyme capable of inducing positive supercoiling, our results suggest the activation of RG activity by UV-induced stress. We suggest simple UV stress could be used to study archaeal DNA repair and responses to DSB.

Enhancement of the photokilling effect of TiO2 in photodynamic therapy by conjugating with reduced graphene oxide and its mechanism exploration.

As a promising next-generation photodynamic therapy (PDT) photosensitizer, TiO2 nanoparticles (NPs) has gained great attention due to its higher efficiency. Yet, its application in PDT is strongly limited by its UV light response range. In this work, TiO2 NPs conjugated with reduced graphene oxide (RGO-TiO2) composites were successfully prepared by hydrothermal reduction method. They were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Transmission electron microscope (TEM), Brunauer-Emmett-Teller (BET), UV-vis spectroscopy and X-ray photoelectron spectroscopy (XPS). Superior adsorption and killing efficiency under UV-A light or visible light were achieved in the presence of the RGO rather than that of unmodified TiO2. The optimal photocatalytic activity was obtained when modified proportion was 0.2 (RGO:TiO2). Dark cytotoxicity was observed using 0-500µgmL-1 RGO-TiO2 during long incubation time. In parallel, following exposure of human hepatocellular carcinoma cell line (HepG2 cells) to RGO-TiO2 and UV-A or visible light irradiation, a marked decrease in the ratio of the super-coiled DNA, mitochondrial membrane potential (MMP), and the oxidative damage effects, as well as increased the apoptosis rate and intracellular calcium concentration were observed. Moreover, photocatalytic RGO-TiO2 composites killed the HepG2 cells by apoptosis pathway. The results suggested that RGO-TiO2 composites were an excellent candidate as a PDT photosensitizer in the near future.

A comprehensive analysis of direct and photosensitized attenuation of Toxoplasma gondii tachyzoites.

In the present work, we have evaluated the effect of three different types of radiation: UVC (254±5nm), UVA (365±20nm) and visible (420±20nm) on different morphological and biological functions of Toxoplasma gondii tachyzoites. Briefly, UVC and UVA showed an inhibitory effect on parasite invasion in a dose-dependent manner. UVC showed the strongest effect inducing both structural damage (antigens) and functional inhibition (i.e., invasion and replication). On its own, visible light induces a quite distinctive and selective pattern of parasite-attenuation. This type of incident radiation inhibits the replication of the parasite affecting neither the capability of invasion/attachment nor the native structure of proteins (antigens) on parasites. Such effects are a consequence of photosensitized processes where phenol red might act as the active photosensitizer. The potential uses of the methodologies investigated herein are discussed.

The water soluble peripherally tetra-substituted zinc(ii), manganese(iii) and copper(ii) phthalocyanines as new potential anticancer agents.

In this study, [2-(2-morpholin-4-ylethoxy)ethoxy] group substituted zinc(ii), manganese(iii) and copper(ii) phthalocyanines 2-4 and their water soluble derivatives 2a, 3a and 4a were synthesized and the interactions of compounds 2a, 3a and 4a with CT-DNA and supercoiled pBR322 plasmid DNA were investigated. The results of binding experiments showed that these compounds were able to interact with CT-DNA via intercalative mode with a strong binding affinity in the order 3a > 2a > 4a. DNA-photocleavage activities of compounds 2a, 3a and 4a were determined. These compounds cleaved supercoiled pBR322 plasmid DNA efficiently under irradiation at 650 nm for 2a and 4a, and at 750 nm for 3a. These compounds displayed remarkable inhibitory activities against topoisomerase I enzyme in a dose-dependent manner. All of these results suggest that these phthalocyanines might be suitable anticancer agents due to their strong binding affinities, significant cleavage activities and effective topoisomerase I inhibition.

Breaking DNA strands by extreme-ultraviolet laser pulses in vacuum.

Ionizing radiation induces a variety of DNA damages including single-strand breaks (SSBs), double-strand breaks (DSBs), abasic sites, modified sugars, and bases. Most theoretical and experimental studies have been focused on DNA strand scissions, in particular production of DNA double-strand breaks. DSBs have been proven to be a key damage at a molecular level responsible for the formation of chromosomal aberrations, leading often to cell death. We have studied the nature of DNA damage induced directly by the pulsed 46.9-nm (26.5 eV) radiation provided by an extreme ultraviolet (XUV) capillary-discharge Ne-like Ar laser (CDL). Doses up to 45 kGy were delivered with a repetition rate of 3 Hz. We studied the dependence of the yield of SSBs and DSBs of a simple model of DNA molecule (pBR322) on the CDL pulse fluence. Agarose gel electrophoresis method was used for determination of both SSB and DSB yields. The action cross sections of the single- and double-strand breaks of pBR322 plasmid DNA in solid state were determined. We observed an increase in the efficiency of strand-break induction in the supercoiled DNA as a function of laser pulse fluence. Results are compared to those acquired at synchrotron radiation facilities and other sources of extreme-ultraviolet and soft x-ray radiation.

Synthesis, X-ray structure and in vitro cytotoxicity studies of Cu(I/II) complexes of thiosemicarbazone: special emphasis on their interactions with DNA.

4-(p-X-phenyl)thiosemicarbazone of napthaldehyde {where X = Cl (HL¹) and X = Br (HL²)}, thiosemicarbazone of quinoline-2-carbaldehyde (HL³) and 4-(p-fluorophenyl)thiosemicarbazone of salicylaldehyde (H2L4) and their copper(I) {[Cu(HL¹)(PPh3)2Br]·CH3CN (1) and [Cu(HL²)(PPh3)2Cl]·DMSO (2)} and copper(II) {[(Cu2L³2Cl)2(µ-Cl)2]·2H2O (3) and [Cu(L4)(Py)] (4)} complexes are reported herein. The synthesized ligands and their copper complexes were successfully characterized by elemental analysis, cyclic voltammetry, NMR, ESI-MS, IR and UV-Vis spectroscopy. Molecular structures of all the Cu(I) and Cu(II) complexes have been determined by X-ray crystallography. All the complexes (1-4) were tested for their ability to exhibit DNA-binding and -cleavage activity. The complexes effectively interact with CT-DNA possibly by groove binding mode, with binding constants ranging from 104 to 105 M⁻¹. Among the complexes, 3 shows the highest chemical (60%) as well as photo-induced (80%) DNA cleavage activity against pUC19 DNA. Finally, the in vitro antiproliferative activity of all the complexes was assayed against the HeLa cell line. Some of the complexes have proved to be as active as the clinical referred drugs, and the greater potency of 3 may be correlated with its aqueous solubility and the presence of the quinonoidal group in the thiosemicarbazone ligand coordinated to the metal.

Seabuckthron (Hippophae rhamnoides L.) leaf extract ameliorates the gamma radiation mediated DNA damage and hepatic alterations.

In vitro assessment showed that H. rhamnoides (HrLE) extract possessed free radical scavenging activities and can protect gamma (gamma) radiation induced supercoiled DNA damage. For in vivo study, Swiss albino mice were administered with HrLE (30 mg/kg body weight) for 15 consecutive days before exposing them to a single dose of 5 Gy of beta radiation. HrLE significantly prevented the radiation induced genomic DNA damage indicated as a significant reduction in the comet parameters. The lipid peroxidation, liver function enzymes, expression of phosphorylated NFkappaB (p65) and IkappaBalpha increased whereas the endogenous antioxidants diminished upon radiation exposure compared to control. Pretreatment of HrLE extract ameliorated these changes. Based on the present results it can be concluded that H. rhamnoides possess a potential preventive element in planned and accidental nuclear exposures.

Non-DSB clustered DNA lesions induced by ionizing radiation are largely responsible for the loss of plasmid DNA functionality in the presence of cisplatin.

The combination of cisplatin and ionizing radiation (IR) increases cell toxicity by both enhancing DNA damage and inhibiting repair mechanisms. Although the formation of cluster DNA lesions, particularly double-strand breaks (DSB) at the site of cisplatin-DNA-adducts has been reported to induce cell death, the contribution of DSB and non-DSB cluster lesions to the cellular toxicity is still unknown. Although both lesions are toxic, it is not always possible to measure their frequency and cell survival in the same model system. To overcome this problem, here, we investigate the effect of cisplatin-adducts on the induction of DSB and non-DSB cluster DNA lesions by IR and determine the impact of such lesions on plasmid functionality. Cluster lesions are two or more lesions on opposite DNA strands with a short distance such that error free repair is difficult or impossible. At a ratio of two cisplatin per plasmid, irradiation of platinated DNA in solution with (137)Cs γ-rays shows enhancements in the formation of DNA DSB and non-DSB cluster lesions by factors of 2.6 and 2.1, respectively, compared to unmodified DNA. However, in absolute terms, the yield for non-DSB cluster lesions is far larger than that for DSB, by a factor of 26. Unmodified and cisplatin-modified DNA were irradiated and subsequently transformed into Escherichia coli to give survival curves representing the functionality of the plasmid DNA as a function of radiation dose. Our results demonstrate that non-DSB cluster lesions are the only toxic lesions present at a sufficient frequency to account for the loss of DNA functionality. Our data also show that Frank-DSB lesions are simply too infrequent to account for the loss of DNA functionality. In conclusion, non-DSB cluster DNA damage is known to be difficult to repair and is probably the lesion responsible for the loss of functionality of DNA modified by cisplatin.

Nuclear aggregates of polyamines in a radiation-induced DNA damage model.

Polyamines (PA) are believed to protect DNA minimizing the effect of radiation damage either by inducing DNA compaction and aggregation or acting as scavengers of free radicals. Using an in vitro pDNA double strand breakage assay based on gel electrophoretic mobility, we compared the protective capability of PA against γ-radiation with that of compounds generated by the supramolecular self-assembly of nuclear polyamines and phosphates, named Nuclear Aggregates of Polyamines (NAPs). Both unassembled PA and in vitro produced NAPs (ivNAPs) were ineffective in conferring pDNA protection at the sub-mM concentration. Single PA showed an appreciable protective effect only at high (mM) concentrations. However, concentrations of spermine (4+) within a critical range (0.481 mM) induced pDNA precipitation, an event that was not observed with NAPs-pDNA interaction. We conclude that the interaction of individual PA is ineffective to assure DNA protection, simultaneously preserving the flexibility and charge density of the double strand. Furthermore, data obtained by testing polyamine and ivNAPS with the current radiation-induced DNA damage model support the concept that PA-phosphate aggregates are the only forms through which PA interact with DNA.

Photoactivated cyclization of aryl-containing enediynes coated gold nanoparticles: enhancement of the DNA cleavage ability of enediynes.

Two novel enediynes containing an aromatic ring and substituted by two thiol functions as end-groups were designed and studied as functionalizing agent of gold nanoparticles. Phototriggered cyclization of the capping agent under UV-visible irradiation was investigated. Interestingly, the length of the thiol-substituted chain was shown to influence significantly the cyclization rate. Depending on the length of the spacer, either polymerization or simple cyclization of the coating agent was evidenced. The present study underscores the possibility of finely controlling the fate of the coating agent (polymerization/cyclization). Nanocomposites were characterized by UV-visible absorption spectroscopy, dynamic light scattering (DLS) technique and transmission electron microscopy (TEM) measurements. Finally, the ability of the colloidal solutions to induce photoinitiated damages to PcDNA3 supercoiled DNA was evaluated. Interestingly, an increase as high as 50% of the DNA cleavage could be registered when adding enediynes-capped gold nanoparticles to solutions of enediynes. In particular, the enhancement of DNA scission was observed in both thermal and photochemical activation modes.

New insights into the mechanism underlying the synergistic action of ionizing radiation with platinum chemotherapeutic drugs: the role of low-energy electrons.

PURPOSE: To investigate the efficiencies of platinum chemotherapeutic drugs (Pt-drugs) in the sensitization of DNA to the direct effects of ionizing radiation and to determine the role of low-energy electrons (LEEs) in this process. METHODS AND MATERIALS: Complexes of supercoiled plasmid DNA covalently bound to either cisplatin, carboplatin, or oxaliplatin were prepared in different molar ratios. Solid films of DNA and DNA modified by Pt-drugs were irradiated with either 10-KeV or 10-eV electrons. Damages to DNA were quantified by gel electrophoresis, and the yields for damage formation were obtained from exposure-response curves. RESULTS: The presence of an average of 2 Pt-drug-DNA adducts (Pt-adducts) in 3199-bp plasmid DNA increases the probability of a double-strand break by factors of 3.1, 2.5, and 2.4 for carboplatin, cisplatin, and oxaliplatin, respectively. Electrons with energies of 10 eV and 10 KeV interact with Pt-adducts to preferentially enhance the formation of cluster lesions. The maximum increase in radiosensitivity per Pt-adduct is found at ratios up to 3.1×10(-4) Pt-adducts per nucleotide, which is equivalent to an average of 2 adducts per plasmid. Carboplatin and oxaliplatin show higher efficiencies than cisplatin in the radiosensitization of DNA. Because carboplatin and cisplatin give rise to identical reactive species that attach to DNA, carboplatin must be considered as a better radiosensitizer for equal numbers of Pt-adducts. CONCLUSION: Platinum chemotherapeutic drugs preferentially enhance the formation of cluster damage to DNA induced by the direct effect of ionizing radiation, and LEEs are the main species responsible for such an enhancement via the formation of electron resonances.

Engineering pH-gated transitions for selective and efficient double-strand DNA photocleavage in hypoxic tumors.

We describe a family of hybrid compounds for the most efficient light-activated double-strand (ds) DNA cleavage known to date. This family represents the second generation of "switchable" molecular systems for pH-gated ds DNA-cleavage which combine a potent DNA-photocleaver and a pH-regulated part derived from a dipeptide. Design of the pH-switchable part utilizes amino groups of different basicity. Whereas the basic amino groups are protonated throughout the biologically relevant pH range, the pH-gating amines undergo protonation at the pH threshold which separates cancer and normal cells. Control over the reactivity and selectivity is achieved via transformation of the initial protonation state (a monocation or a dication) into a trication at the acidic pH. This change leads to an extraordinary increase in the efficiency of ds DNA cleavage leading to the ds:ss ratios comparable with the most efficient nonenzymatic ds DNA cleavers. Statistical analysis reveals that these high ds:ss ratios result from the combination of several factors: (a) true double-stranded cleavage, and (b) conversion of single-stranded (ss)-scission into ds cleavage. Considerable part of ds cleavage is also produced via the combination of ss cleavage events.

Effects of X-irradiation on mitochondrial DNA damage and its supercoiling formation change.

There have been a small number of reports of radiation-induced mtDNA damage, and mtDNA supercoiling formation change induced by ionizing radiation has not been investigated before. This study evaluated mtDNA damage and supercoiling formation change after X-irradiation. The human breast cancer cell line, MCF-7 cells were used for analysis. Modified supercoiling-sensitive real-time PCR approach was used to evaluate mitochondrial DNA supercoiling formation change and copy number; long-PCR method was applied for the quantification of mtDNA damage. MtDNA damage and formation change induced by high-dose irradiation was persistent in 24h after irradiation and was not significant after low-dose irradiation. MtDNA copy number was slightly increased after high-dose irradiation and a transit increase was observed after low-dose irradiation. This is the first study to evaluate radiation-induced mitochondrial DNA supercoiling formation change using real-time PCR. Combined with data of ROS generation and dynamics of mitochondrial mass, our findings suggested that mtDNA is sensitive to radiation hazards, indicating mitochondrial biogenesis play an important role in radiation-induced cellular response.

Novel method for quantifying radiation-induced single-strand-break yields in plasmid DNA highlights 10-fold discrepancy.

The widely used agarose gel electrophoresis method for assessing radiation-induced single-strand-break (SSB) yield in plasmid DNA involves measurement of the fraction of relaxed-circular (C) form that migrates independently from the intact supercoiled (SC) form. We rationalized that this method may underestimate the SSB yield since the position of the relaxed-circular form is not altered when the number of SSB per DNA molecule is >1. To overcome this limitation, we have developed a novel method that directly probes and quantifies SSBs. Supercoiled (3)H-pUC19 plasmid samples were irradiated with γ-rays, alkali-denatured, dephosphorylated, and kinated with γ-[(32)P]ATP, and the DNA-incorporated (32)P activities were used to quantify the SSB yields per DNA molecule, employing a standard curve generated using DNA molecules containing a known number of SSBs. The same irradiated samples were analyzed by agarose gel and SSB yields were determined by conventional methods. Comparison of the data demonstrated that the mean SSB yield per plasmid DNA molecule of [21.2±0.59]×10(-2)Gy(-1) as measured by direct probing is ~10-fold higher than that obtained from conventional gel-based methods. These findings imply that the SSB yields inferred from agarose gels need reevaluation, especially when they were utilized in the determination of radiation risk.

Cysteine-rich cyanopeptide beta2 from Spirulina fusiformis exhibits plasmid DNA pBR322 scission prevention and cellular antioxidant activity.

Isolation of three different active peptides from C-phycocyanin (C-pc) beta chain of S. fusiformis and their biological properties are reported. Phycocyanin peptide beta fraction 2 (cyanopeptide beta 2) facilitated both antioxidant and plasmid DNA strand scission prevention activity due to higher cysteine moieties in the isolated peptide. The peptide significantly scavenged the free radicals like 1-1,-diphenyl-2-picryl hydrazyl and ferric reducing ability of plasma, increased the absorbance values in reducing power and also showed the higher trolox equivalent antioxidant capacity values in total reactive antioxidant potentials assay. Cyanopeptide beta 2 also inhibited reactive oxygen species induced DNA pBR322 damage in dose dependent manner along with free radical scavenging properties suggesting the role in the DNA integrity which is also evident by DNA binding activity of peptide. In addition, the generation of reactive oxygen species (ROS) was dose dependent (10 and 20 ng/ml) and significantly quenched by cyanopeptide beta2 in human fibroblast cell line TIG 3-20. In vitro cell scratch injury assay demonstrated the capacity of cyanopeptide beta2 in cell migration in to wounded area suggesting fibroblast proliferation and migration. The results suggest that cyanopeptide beta2 can be a free radical scavenger and effective peptide for future biomedical applications like wound healing, atherosclerosis, cell redox potential and hypoxia.

Platinum nanoparticles: a promising material for future cancer therapy?

Recently, the use of gold nanoparticles as potential tumor selective radiosensitizers has been proposed as a breakthrough in radiotherapy. Experiments in living cells and in vivo have demonstrated the efficiency of the metal nanoparticles when combined with low energy x-ray radiations (below conventional 1 MeV Linac radiation). Further studies on DNA have been performed in order to better understand the fundamental processes of sensitization and to further improve the method. In this work, we propose a new strategy based on the combination of platinum nanoparticles with irradiation by fast ions effectively used in hadron therapy. It is observed in particular that nanoparticles enhance strongly lethal damage in DNA, with an efficiency factor close to 2 for double strand breaks. In order to disentangle the effect of the nano-design architecture, a comparison with the effects of dispersed metal atoms at the same concentration has been performed. It is thus shown that the sensitization in nanoparticles is enhanced due to auto-amplified electronic cascades inside the nanoparticles, which reinforces the energy deposition in the close vicinity of the metal. Finally, the combination of fast ion radiation (hadron therapy) with platinum nanoparticles should strongly improve cancer therapy protocols.

Gold nanoparticles enhance DNA damage induced by anti-cancer drugs and radiation.

The chemotherapeutic agent cisplatin was chemically linked to pGEM-3Zf(-) plasmid DNA to produce a cisplatin-DNA complex, Gold nanoparticles, which bind electrostatically to pure DNA, could also be added to this complex. Dry films of pure plasmid DNA and DNA-cisplatin, DNA-gold nanoparticles and DNA-cisplatin-gold nanoparticles complexes were bombarded by 60 keV electrons. The yields of single- and double-strand breaks were measured as a function of exposure by electrophoresis. From a comparison of such yields from the different type of films, we found that the binding of only one gold nanoparticle to a plasmid-cisplatin complex containing 3197 base pairs increases by a factor of 3 the efficiency of the chemotherapeutic agent cisplatin to produce double-strand breaks in irradiated DNA. Furthermore, adding two cisplatin molecules and one gold nanoparticle to DNA enhances radiation-induced DSBs by a factor of 7.5. A number of phenomena could contribute to this huge enhancement, including the higher density of low-energy electrons and reactive species around the gold nanoparticles and the weakening of bonds adjacent to cisplatin in the DNA backbone. The addition of gold nanoparticles to cisplatin and other platinum agents may therefore provide interesting avenues of research to improve the treatment of cancer by concomitant chemoradiation.

Novel methodology for predicting photogenotoxic risk of pharmaceutical substances based on reactive oxygen species (ROS) and DNA-binding assay.

Drug-induced phototoxic skin responses involves photoirritation, photoallergy, and photogenotoxicity. In the present study, we attempted to develop effective analytical tools to predict the photogenotoxic potential of pharmaceutical substances on the basis of their photobiological and DNA-binding properties. Photocleavage of plasmid DNA was assessed by capillary gel electrophoresis (CGE), and generation of reactive oxygen species (ROS) from photoirradiated photosensitizers was also assessed by spectrophotometrical determination. The affinity of drugs with DNA was determined by fluorescent titration or competitive binding assay using ethidium bromide. Structural transition from supercoiled form to open circular form of plasmid DNA were observed in the presence of nine irradiated-photosensitizers, however DNA damage by six phototoxic and five nonphototoxic drugs were negligible or weak even though some chemicals exhibited significant generation of ROS. DNA photocleavers tended to interact with DNA, binding constants of which were found to be less than 1 microM, whereas most nonphotogenotoxic chemicals failed to bind with DNA. The differences in DNA-binding properties might be attributed to a data discrepancy between ROS-generation and DNA photocleaving activity. The combination use of ROS assay and DNA-binding assay could be fast and reliable prediction for the photogenotoxic potential of a large number of drug candidates at the drug-discovery stage.

A radiation target method for size determination of supercoiled plasmid DNA.

Supercoiled DNA plasmids were exposed in the frozen state to high-energy electrons. Surviving supercoiled molecules were separated from their degradation products (e.g., open circle and linear forms) by agarose gel electrophoresis and subsequently quantified by staining and image analysis. Complex survival curves were analyzed using radiation target theory, yielding the radiation-sensitive mass of each form. One of the irradiated plasmids was transfected into cells, permitting radiation analysis of gene expression. Loss of this function was associated with a mass much smaller than the entire plasmid molecule, indicating a lack of energy transfer in amounts sufficient to cause structural damage along the DNA polynucleotide. The method of radiation target analysis can be applied to study both structure and function of DNA.

DNA double-strand breaks induced by decay of (123)I-labeled Hoechst 33342: role of DNA topology.

PURPOSE: To determine double-strand-break (DSB) yields produced by decay of minor-groove-bound (123)I-labeled Hoechst 33342 ((123)IEH) in supercoiled (SC) and linear (L) forms of pUC19 DNA, to compare strand-break efficiency of (123)IEH with that of (125)IEH, and to examine the role of DNA topology in DSB induction by these Auger electron emitters. MATERIALS AND METHODS: Tritium-labeled SC and L pUC19 DNA were incubated with (123)IEH (0-10.9 MBq) at 4 degrees C. After (123)I had completely decayed (10 days), samples were analyzed on agarose gel, and single-strand-break (SSB) and DSB yields were measured. RESULTS: Each (123)I decay in SC DNA produces a DSB yield of 0.18 +/- 0.01. On the basis of DSB yields for (125)IEH (0.52 +/- 0.02 for SC and 1.62 +/- 0.07 for L, reported previously) and dosimetric expectations, a DSB yield of approximately 0.5 (3 x 0.18) per (123)I decay is expected for L DNA. However, no DSB are observed for the L form, even after approximately 2 x 10(11) decays of (123)I per microg DNA, whereas a similar number of (125)I decays produces DSB in approximately 40% of L DNA. CONCLUSION: (123)IEH-induced DSB yield for SC but not L DNA is consistent with the dosimetric expectations for Auger electron emitters. These studies highlight the role of DNA topology in DSB production by Auger emitters and underscore the failure of current theoretical dosimetric methods per se to predict the magnitude of DSB.