Proximity Estimation and Quantification of Ionizing Radiation-induced DNA Lesions in Aqueous Media using Fluorescence Spectroscopy.

Clustered DNA damage (cluster) or a multiply damaged site, which is a region with two or more lesions within one or two helical turns, has a high mutagenic potential and causes cell death. We quantified fluorophore-labeled lesions and estimated their proximity through fluorescence anisotropy measurements depending on Förster resonance energy transfer (FRET) among the fluorophores close to each other. pUC19 plasmid DNA (2,686 base pairs) dissolved in water or 0.2 M Tris-HCl buffer at a concentration of 10 µg/µL was irradiated by several ionizing radiations with varying linear energy transfers (LET, 0.2-1890 keV/µm). Electrophilic carbonyls (aldehydes and ketones) at abasic sites (APs) produced in DNA were labeled with Alexa Fluor 488 fluorescent dyes with an O-amino functional group. Regardless of the presence or absence of the buffer, AP yields (the number of APs/base pair/Gy) tended to decrease with increasing LET, and the ratio of the AP yield (in 0.2 M Tris-HCl/in water) was less than 0.1 in the LET range of 0.2-200 keV/µm. However, in a higher LET range, the ratios were greater than 0.1. At a low dose, fluorescence anisotropy decreased with increasing LET in 0.2 M Tris-HCl, whereas, in water, this LET dependence was almost insignificant. These findings suggest that 1. the damage distribution on a DNA molecule formed by indirect effects (e.g., by hydroxyl radicals) does not depend on radiation quality and 2. greater LET radiation is more likely to produce a cluster and/or to produce a cluster with shorter distances between lesions by direct effects. This FRET-based proximity estimation of DNA lesions will contribute not only to the identification of clusters and their complexity in a whole genome, but also to the study of their repair mechanism by single-molecular level fluorescence microscopy.

A step-by-step simulation code for estimating yields of water radiolysis species based on electron track-structure mode in the PHITS code.

Objective. Time-dependent yields of chemical products resulting from water radiolysis play a great role in evaluating DNA damage response after exposure to ionizing radiation. Particle and Heavy Ion Transport code System (PHITS) is a general-purpose Monte Carlo simulation code for radiation transport, which simulates atomic interactions originating from discrete energy levels of ionizations and electronic excitations as well as molecular excitations as physical stages. However, no chemical code for simulating water radiolysis products exists in the PHITS package.Approach.Here, we developed a chemical simulation code dedicated to the PHITS code, hereafter calledPHITS-Chemcode, which enables the calculation of theGvalues of water radiolysis species (•OH, eaq-, H2, H2O2etc) by electron beams.Main results.The estimatedGvalues during 1 µs are in agreement with the experimental ones and other simulations. ThisPHITS-Chemcode also simulates the radiolysis in the presence of OH radical scavengers, such as tris(hydroxymethyl)aminomethane and dimethyl sulfoxide. Thank to this feature, the contributions of direct and indirect effects on DNA damage induction under various scavenging capacities can be analyzed.Significance.This chemical code coupled with PHITS could contribute to elucidating the mechanism of radiation effects by connecting physical, physicochemical, and chemical processes.

Synthetic DNA binders for fluorescent sensing of thymine glycol-containing DNA duplexes and inhibition of endonuclease activity.

Dimethyllumazine (DML)-thiazole orange (TO) conjugates were developed for fluorescence sensing of thymine glycol (Tg)-containing DNAs based on the selective recognition of the A nucleobase opposite the Tg residue. Additionally, this conjugate has demonstrated an inhibitory activity towards endonuclease III, a DNA repair enzyme, through its competitive binding to Tg-containing DNAs.

Formation of clustered DNA damage in vivo upon irradiation with ionizing radiation: Visualization and analysis with atomic force microscopy.

SignificanceDNA damage causes loss of or alterations in genetic information, resulting in cell death or mutations. Ionizing radiations produce local, multiple DNA damage sites called clustered DNA damage. In this study, a complete protocol was established to analyze the damage complexity of clustered DNA damage, wherein damage-containing genomic DNA fragments were selectively concentrated via pulldown, and clustered DNA damage was visualized by atomic force microscopy. It was found that X-rays and Fe ion beams caused clustered DNA damage. Fe ion beams also produced clustered DNA damage with high complexity. Fe ion beam-induced complex DNA double-strand breaks (DSBs) containing one or more base lesion(s) near the DSB end were refractory to repair, implying their lethal effects.

Fluorescence anisotropy study of radiation-induced DNA damage clustering based on FRET.

A clustered DNA damage site (cluster), in which two or more lesions exist within a few helical turns, is believed to be a key factor determining the fate of a living cell exposed to a DNA damaging agent such as ionizing radiation. However, the structural details of a cluster such as the number of included lesions and their proximity are unknown. Herein, we develop a method to characterize a cluster by fluorescence anisotropy measurements based on Förster resonance energy transfer (homo-FRET). Plasmid DNA (pUC19) was irradiated with 2.0 and 0.52 MeV/u 4He2+, or 0.37 MeV/u 12C5+ ion beams (linear energy transfer: ~ 70, ~ 150, ~ 760 keV/µm, respectively) and 60Co γ-rays as a standard (~ 0.2 keV/µm) in the solid state. The irradiated DNA was labeled with an aminooxyl fluorophore (Alexa Fluor 488) to the aldehyde/ketone moieties such as apurinic/apyrimidinic sites. Homo-FRET analyses provided the apparent base separation values between lesions in a cluster produced by each ion beam track as 21.1, 19.4, and 18.7 base pairs. The production frequency of a cluster increases with increasing linear energy transfer of radiation. Our results demonstrate that homo-FRET analysis has the potential to discover the qualitative and the quantitative differences of the clusters produced not only by a variety of ionizing radiation but also by other DNA damaging agents.

Strand with mutagenic lesion is preferentially used as a template in the region of a bi-stranded clustered DNA damage site in Escherichia coli.

The damaging potential of ionizing radiation arises largely from the generation of clustered DNA damage sites within cells. Previous studies using synthetic DNA lesions have demonstrated that models of clustered DNA damage exhibit enhanced mutagenic potential of the comprising lesions. However, little is known regarding the processes that lead to mutations in these sites, apart from the fact that base excision repair of lesions within the cluster is compromised. Unique features of the mutation frequencies within bi-stranded clusters have led researchers to speculate that the strand containing the mutagenic lesion is preferentially used as the template for DNA synthesis. To gain further insights into the processing of clustered DNA damage sites, we used a plasmid-based assay in E. coli cells. Our findings revealed that the strand containing a mutagenic lesion within a bi-stranded clustered DNA damage site is frequently used as the template. This suggests the presence of an, as yet unknown, strand synthesis process that is unrelated to base excision repair, and that this process plays an important role in mutagenesis. The length of the region of strand preference was found to be determined by DNA polymerase I.

A Simplified Cluster Analysis of Electron Track Structure for Estimating Complex DNA Damage Yields.

Complex DNA damage, defined as at least two vicinal lesions within 10-20 base pairs (bp), induced after exposure to ionizing radiation, is recognized as fatal damage to human tissue. Due to the difficulty of directly measuring the aggregation of DNA damage at the nano-meter scale, many cluster analyses of inelastic interactions based on Monte Carlo simulation for radiation track structure in liquid water have been conducted to evaluate DNA damage. Meanwhile, the experimental technique to detect complex DNA damage has evolved in recent decades, so both approaches with simulation and experiment get used for investigating complex DNA damage. During this study, we propose a simplified cluster analysis of ionization and electronic excitation events within 10 bp based on track structure for estimating complex DNA damage yields for electron and X-ray irradiations. We then compare the computational results with the experimental complex DNA damage coupled with base damage (BD) measured by enzymatic cleavage and atomic force microscopy (AFM). The computational results agree well with experimental fractions of complex damage yields, i.e., single and double strand breaks (SSBs, DSBs) and complex BD, when the yield ratio of BD/SSB is assumed to be 1.3. Considering the comparison of complex DSB yields, i.e., DSB + BD and DSB + 2BD, between simulation and experimental data, we find that the aggregation degree of the events along electron tracks reflects the complexity of induced DNA damage, showing 43.5% of DSB induced after 70 kVp X-ray irradiation can be classified as a complex form coupled with BD. The present simulation enables us to quantify the type of complex damage which cannot be measured through in vitro experiments and helps us to interpret the experimental detection efficiency for complex BD measured by AFM. This simple model for estimating complex DNA damage yields contributes to the precise understanding of the DNA damage complexity induced after X-ray and electron irradiations.

Mutagenic potential of 8-oxo-7,8-dihydroguanine (8-oxoG) is influenced by nearby clustered lesions.

Ionizing radiation causes various different types of DNA damage. If not repaired, DNA damage can have detrimental effects. Previous studies indicate that the spatial distribution of DNA lesions induced by ionizing radiation is highly relevant to the ensuing biological effects. Clustered DNA damage, consisting of DNA lesions in close proximity, has been studied in detail, and has enhanced mutagenic potential depending on the configuration of the lesions. However, it is not known whether clustered DNA damage affects the mutagenic potential of a sufficiently separated, isolated lesion. Using synthetic damage constructs, we investigated the mutagenic potential of an isolated 8-oxo-7,8-dihydroguanine (8-oxoG) separated by at least 7 bp from other lesions. Under the spatial distribution of DNA lesions tested in the present study, neighboring clustered DNA lesions likely retarded the processing of the isolated 8-oxoG and resulted in enhanced mutation frequency. However, the enhanced mutagenic potential was dependent on which strand the isolated 8-oxoG was located. Our results indicate that the processing of a bi-stranded cluster could affect the mutagenic outcome of a nearby isolated lesion, separated up to ∼20 bp.

New method for estimating clustering of DNA lesions induced by physical/chemical mutagens using fluorescence anisotropy.

We have developed a new method for estimating the localization of DNA damage such as apurinic/apyrimidinic sites (APs) on DNA using fluorescence anisotropy. This method is aimed at characterizing clustered DNA damage produced by DNA-damaging agents such as ionizing radiation and genotoxic chemicals. A fluorescent probe with an aminooxy group (AlexaFluor488) was used to label APs. We prepared a pUC19 plasmid with APs by heating under acidic conditions as a model for damaged DNA, and subsequently labeled the APs. We found that the observed fluorescence anisotropy (robs) decreases as averaged AP density (λAP: number of APs per base pair) increases due to homo-FRET, and that the APs were randomly distributed. We applied this method to three DNA-damaging agents, 60Co γ-rays, methyl methanesulfonate (MMS), and neocarzinostatin (NCS). We found that robs-λAP relationships differed significantly between MMS and NCS. At low AP density (λAP < 0.001), the APs induced by MMS seemed to not be closely distributed, whereas those induced by NCS were remarkably clustered. In contrast, the AP clustering induced by 60Co γ-rays was similar to, but potentially more likely to occur than, random distribution. This simple method can be used to estimate mutagenicity of ionizing radiation and genotoxic chemicals.

A polymerization-based method to construct a plasmid containing clustered DNA damage and a mismatch.

Exposure of biological materials to ionizing radiation often induces clustered DNA damage. The mutagenicity of clustered DNA damage can be analyzed with plasmids carrying a clustered DNA damage site, in which the strand bias of a replicating plasmid (i.e., the degree to which each of the two strands of the plasmid are used as the template for replication of the plasmid) can help to clarify how clustered DNA damage enhances the mutagenic potential of comprising lesions. Placement of a mismatch near a clustered DNA damage site can help to determine the strand bias, but present plasmid-based methods do not allow insertion of a mismatch at a given site in the plasmid. Here, we describe a polymerization-based method for constructing a plasmid containing clustered DNA lesions and a mismatch. The presence of a DNA lesion and a mismatch in the plasmid was verified by enzymatic treatment and by determining the relative abundance of the progeny plasmids derived from each of the two strands of the plasmid.

Localization estimation of ionizing radiation-induced abasic sites in DNA in the solid state using fluorescence resonance energy transfer.

Clustered DNA damage is considered an important factor in determining the biological consequences of ionizing radiation. In this study, we successfully estimated the localization of abasic sites (APs) in DNA exposed to ionizing radiation using fluorescence resonance energy transfer (FRET) without any involvement of repair enzyme functions. A linearized plasmid (pUC19 digested by Sma I) was irradiated with: (60)Co γ rays; (4)He(2+) (2.0 MeV/u) particles; and the (12)C(5+) (0.37 MeV/u) particles in the solid state. A donor or acceptor fluorescent probe with a nucleophilic O-amino group was used to label APs. The results showed that the (12)C(5+) particle likely produced close APs within a track. The apparent distance calculated from the observed FRET efficiency (E) of around 0.10 was estimated to be approximately 17 base pairs. On the other hand, E values of (60)Co γ rays and the (4)He(2+) beam were less than those of the (12)C(5+) beam, increased with increasing AP density (the average number of APs per base pair), and were slightly greater than those of randomly distributed APs. We propose that the FRET method provides a degree of localization regardless of whether an AP cluster is single-stranded or bistranded DNA damage.

Significance of DNA polymerase I in in vivo processing of clustered DNA damage.

We examined the biological consequences of bi-stranded clustered damage sites, consisting of a combination of DNA lesions, such as a 1-nucleotide gap (GAP), an apurinic/apyrimidinic (AP) site, and an 8-oxo-7,8-dihydroguanine (8-oxoG), using a bacterial plasmid-based assay. Following transformation with the plasmid containing bi-stranded clustered damage sites into the wild type strain of Escherichia coli, transformation frequencies were significantly lower for the bi-stranded clustered GAP/AP lesions (separated by 1bp) than for either a single GAP or a single AP site. When the two lesions were separated by 10-20bp, the transformation efficiencies were comparable with those of the single lesions. This recovery of transformation efficiency for separated lesions requires DNA polymerase I (Pol I) activity. Analogously, the mutation frequency was found to depend on the distance separating lesions in a bi-stranded cluster containing a GAP and an 8-oxoG, and Pol I was found to play an important role in minimising mutations induced as a result of clustered lesions. The mutagenic potential of 8-oxoG within the bi-stranded lesions does not depend on whether it is situated on the leading or lagging strand. These results indicate that the biological consequences of clustered DNA damage strongly depend on the extent of repair of the strand breaks as well as the DNA polymerase in lesion-avoidance pathways during replication.

Development of 'leaky' liposome triggered by radiation applicable to a drug reservoir and a simple radiation dosimeter.

Radiation effects on permeability of liposomes with different lipid compositions and the content ratios were investigated using X-rays. Liposomes constituted from an unsaturated phosphatidylcholine (PC), a saturated PC, and cholesterol were selected. As a result, we found that a PC with bis-allylic hydrogen, particularly dilinoleoyl PC (DLOPC), is essential for the radiation sensitivity in permeability, and it can be controlled by changing DLOPC content and dose rate. These characteristics are contributive to a novel pharmaceutical and a new-conceptual radiation dosimeter.

A methodology for estimating localization of apurinic/apyrimidinic sites in DNA using fluorescence resonance energy transfer.

We have developed a methodology for estimating localization of lesions on double-stranded DNA using fluorescence resonance energy transfer (FRET). We focused on apurinic/apyrimidinic (AP) sites, which are typical DNA lesions induced by radiation and chemicals and produced spontaneously under physiological conditions. Donor-acceptor fluorescent probes with O-amino groups (Alexa Fluor 350-Alexa Fluor 488 dye pair) were used for selectively labeling AP sites. pUC19 plasmid subjected to heat treatment (pH 5.2, 70 °C) was used as a model double-stranded DNA containing AP sites. The results of both FRET analysis and theoretical study enabled us to prove that AP sites induced by the heat treatment are distributed almost randomly along the DNA molecule. This methodology will be useful for estimating the risk of ionizing radiation and chemicals (e.g., pollutants and anticancer agents) based on the probability of producing "clustered DNA damage sites," which are considered to be less easily repairable and, therefore, more harmful to living systems.

Characterization of lesions induced in linear-formed plasmid DNA by valence ionization and Auger decay at carbon, nitrogen and oxygen.

PURPOSE: To study the DNA lesions induced by the Auger decay of carbon, nitrogen, and oxygen using ultrasoft X-rays (USX) that are expected to be important with the DNA repair system of living cells. MATERIALS AND METHODS: pUC19 plasmid DNA dry samples were irradiated with USX photons at 270 and 560 eV and (60)Co gamma-rays in vacuum at room temperature. The amounts of unaltered base release by the direct radiation effects were quantified using high-performance liquid chromatography. To quantify and characterize the strand break termini the rate at which snake venom phosphodiesterase (SVPD) digested irradiated DNA pretreated with and without calf intestine alkaline phosphatase was measured. Moreover, the piperidine-labile base lesions and abasic sites of the irradiated DNA were estimated using the SVPD method. RESULTS: The yields of unaltered base release for 270, 560 eV photons and (60)Co gamma-rays were 0.016, 0.014, and 0.018 micromol/J, respectively. The total 3' termini for the three kinds of photons were around 0.1 micromol/J. The production of 3' termini with phosphate was found to be predominant with respect to that of 3'OH termini for the three kinds of radiation. The yield of piperidine-labile sites for 270 eV ( approximately 0.1 micromol/J) was slightly larger than that for 560 eV ( approximately 0.07 micromol/J) and also for gamma-rays ( approximately 0.082 micromol/J). CONCLUSION: Although the Auger process in DNA-constituent atoms was expected to induce Auger-specific lesions in the molecule the chemical endpoints would have been covered with a large number of lesions produced from secondary electrons in the surrounding bulk DNA molecules. The present results, however, suggest that a low-energy electron field produced by the USX photons in the bulk DNA is basically not at all specific to DNA damage being produced when compared with the high-energy electron field produced by (60)Co gamma-rays.

A novel methodology for characterizing strand-break termini and damaged bases in plasmid DNA exposed to ionizing radiation.

We have developed a de novo methodology to characterize radiation damage in DNA. An enzyme system consisting of the 3'-->5' exonuclease snake venom phosphodiesterase (SVPD) and calf intestine alkaline phosphatase (CIAP) was used to examine the 3' termini of strand-break sites. In this study, we hypothesized that the strand-break termini can be divided into two categories: CIAP-independent SVPD sites and CIAP-dependent SVPD sites. The former consists of strand-break termini that can be recognized directly and digested by SVPD without CIAP pretreatment, whereas the latter includes the termini that cannot be digested by SVPD without CIAP pretreatment. In addition, the apparent radiation-chemical yield (G value) can be estimated using the level of intact 2'-deoxynucleotides produced during a 15-min incubation with SVPD. The G value for total strand breaks in fully dried DNA irradiated with (60)Co gamma-rays was estimated to be 0.1 micromol/J. Moreover, the G values of CIAP-dependent and CIAP-independent SVPD sites were estimated to be 0.078 and 0.024 micromol/J, respectively. These values suggest that 3'-phosphate termini are more likely to be produced than 3' termini without phosphate. Furthermore, piperidine-treated irradiated plasmid DNA was also treated with the same enzyme system to examine the piperidine-labile sites. As a result of the treatment, the G value of the CIAP-dependent SVPD sites increased to 0.16 micromol/J, whereas no significant increase was seen in the G value of the CIAP-independent SVPD sites. This observation implies that most piperidine-labile damaged bases can be eliminated to form apurinic/apyrimidinic sites, which are completely removed by piperidine treatment to form 3' phosphate termini, and that prompt CIAP-independent SVPD sites are piperidine resistant.

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.

Anti-interleukin 6 receptor antibody inhibits murine AA-amyloidosis.

OBJECTIVE: AA-amyloidosis is a severe complication in chronic inflammatory diseases. AA-amyloidosis is caused by the deposition of insoluble fibrils containing AA amyloid protein derived from serum amyloid A (SAA), which is synthesized by inflammatory cytokine stimulation. We examined whether anti-interleukin 6 receptor (IL-6R) antibody prevented the development of AA-amyloidosis in mouse models. METHODS: A transient model was induced by the injection of amyloid enhancing factor (AEF) and adjuvant treatment in C57BL/6 mice. Monoclonal IgG1 antibody, MR16-1, was injected intraperitoneally just once before the injection of AEF and adjuvant. After 2 and 5 weeks, mice were sacrificed and histologically examined. In contrast, a chronic model was induced by AEF injection into IL-6 transgenic mice. One week later, in order to avoid neutralizing antibody production, MR16-1 (200 mg/kg) was injected intravenously. MR16-1 (5 mg/kg) was injected subcutaneously twice a week from the next week. Three and 6 weeks after AEF injection, mice were sacrificed and histologically examined. RESULTS: In the transient model, amyloid deposition was observed in the spleen, liver, and kidney as early as 2 weeks after treatment. MR16-1 completely prevented amyloid deposition. Although IL-6 production was not suppressed, SAA production was significantly suppressed. In the chronic model, substantial amyloid deposition was seen in multiple organs and tissues as well as the spleen, liver, and kidney. MR16-1 suppressed amyloid deposition in many organs, even when injected one week after AEF injection; it showed a tendency to decrease SAA and IL-6 levels were decreased. CONCLUSION: IL-6 is a key cytokine for the induction of AA-amyloidosis, and anti-IL-6R therapy appears promising for the treatment of AA-amyloidosis.

Qualitative and quantitative analyses of the decomposition products that arise from the exposure of thymine to monochromatic ultrasoft X rays and 60Co gamma rays in the solid state.

HPLC analyses of condensed thymine irradiated with monochromatic synchrotron ultrasoft X rays in the energy region around nitrogen and oxygen K-shell edges were performed. Cobalt-60 gamma rays were used as a reference radiation. The radiation chemical dose response of each separated thymine decomposition product was also determined. Uracil (U), 5-(hydroxymethyl)uracil (HMU), 5,6-dihydrothymine (DHT), 5-formyluracil (foU) and four main unknown products were found in the HPLC chromatogram of the sample irradiated with ultrasoft X rays in vacuo. Similar spectra of the products were also found in the gamma-ray experiment; however, some unknown products that appeared after elution of the thymine peak were significantly larger than those in the ultrasoft X- ray experiment. This result indicates the difference in radiation quality. The G value of DHT produced by gamma radiation was 10 times larger than those produced by the ultrasoft X- ray photons with energies of 395 and 407 eV corresponding to below and on the nitrogen K-shell edge, respectively. This result suggests that the differences in the photon energy and/ or in the energy spectra of the secondary electron between ultrasoft X rays and gamma rays are causing differences in the process of the radiation chemistry. Moreover, the yields of all the thymine decomposition products induced by 538 eV photons (oxygen K-shell edge) were significantly smaller than those induced by photons around the nitrogen K-shell edge. The K-shell excitation of oxygen in thymine may efficiently promote the production of small thymine fragments susceptible to desorption from the sample.