Ultrafast fragmentation of highly-excited doubly-ionized deoxyribose: role of the liquid water environment.

Ab initio molecular dynamics simulations are used to investigate the fragmentation dynamics following the double ionization of 2-deoxy-D-ribose (DR), a major component in the DNA chain. Different ionization scenarios are considered to provide a complete picture. First focusing on isolated DR2+, fragmentation patterns are determined for the ground electronic state, adding randomly distributed excitation energy to the nuclei. These patterns differ for the two isomers studied. To compare thermal and electronic excitation effects, Ehrenfest dynamics are also performed, allowing to remove the two electrons from selected molecular orbitals. Two intermediate-energy orbitals, localized on the carbon chain, were selected. The dissociation pattern corresponds to the most frequent pattern obtained when adding thermal excitation. On the contrary, targeting the four deepest orbitals, localized on the oxygen atoms, leads to selective ultrafast C-O and/or O-H bond dissociation. To probe the role of environment, a system consisting of a DR molecule embedded in liquid water is then studied. The two electrons are removed from either the DR or the water molecules directly linked to the sugar through hydrogen bonds. Although the dynamics onset is similar to that of isolated DR when removing the same deep orbitals localized on the sugar oxygen atoms, the subsequent fragmentation patterns differ. Sugar damage also occurs following the Coulomb explosion of neighboring H2O2+ molecules due to interaction with the emitted O or H atoms.

Comparison of core and valence band electronic structures of bulk uracil and 5-halouracils.

Core (C, N, and O 1s regions) and valence band electronic structures of bulk uracil and 5-fluoro-, -chloro-, and -iodouracils were investigated using X-ray photoemission spectroscopy and comprehensively compared with those of 5-bromouracil measured under the same experimental conditions before. The halogenation of uracil shifted the core peaks of the 5-position carbons toward the higher binding energy side and reduced the ionization potentials depending on the type of halogen. Theoretical calculations supported these results. The alterations of electronic properties induced by the halogenation would result in the characteristic properties of 5-halouracils.

First-principles simulation of an ejected electron produced by monochromatic deposition energy to water at the femtosecond order.

This study uses a time-dependent first-principles simulation code to investigate the transient dynamics of an ejected electron produced in the monochromatic deposition energy from 11 to 19 eV in water. The energy deposition forms a three-body single spur comprising a hydroxyl radical (OH˙), hydronium ion (H3O+), and hydrated electron (eaq-). The earliest formation involves electron thermalization and delocalization dominated by the molecular excitation of water. Our simulation results show that the transient electron dynamics primarily depends on the amount of deposition energy to water; the thermalization time varies from 200 to 500 fs, and the delocalization varies from 3 to 10 nm in this energy range. These features are crucial for determining the earliest single-spur formation and facilitating a sequential simulation from an energy deposition to a chemical reaction in water photolysis or radiolysis. The spur radius obtained from the simulation correlates reasonably with the experimental-based estimations. Our results should provide universalistic insights for analysing ultrafast phenomena dominated by the molecular excitation of water in the femtosecond order.

Temporal Dynamic Regulation of Autophagy and Senescence Induction in Response to Radiation Exposure.

Autophagy and senescence are closely related cellular responses to genotoxic stress, and play significant roles in the execution of cellular responses to radiation exposure. However, little is known about their interplay in the fate-decision of cells receiving lethal doses of radiation. Here, we report that autophagy precedes the establishment of premature senescence in normal human fibroblasts exposed to lethal doses of radiation. Activation of the p53-dependent DNA damage response caused sustained dephosphorylation of RB proteins and consequent cell cycle arrest, concurrently with Ulk1 dephosphorylation at Ser638 by PPM1D, which promoted autophagy induction 1-2 days after irradiation. In addition, mitochondrial fragmentation became obvious 1-2 days after irradiation, and autophagy was further enhanced. However, Ulk1 levels decreased significantly after 2 days, resulting in lower LC3-II levels. An autophagic flux assay using chloroquine (CQ) also revealed that the flux in irradiated cells gradually decreased over 30 days. In contrast, lysosomal augmentation started at 1 day, became significantly upregulated after 5 days, and continued for over 30 days. After a rapid decrease in autophagy, p16 expression increased and senescence was established, but autophagic activity remained reduced. These results demonstrated that X-ray irradiation triggered two processes, autophagy and senescence, with the former being temporary and regulated by DNA damage response and mitophagy, and the latter being sustained and regulated by persistent cell cycle arrest. The interplay between autophagy and senescence seems to be essential for the proper implementation of the cellular response to radiation exposure.

Structural study of wild-type and phospho-mimic XRCC4 dimer and multimer proteins using circular dichroism spectroscopy.

PURPOSE: To investigate the structural features of wild-type and phospho-mimicking mutated XRCC4 protein, a protein involved in DNA double-strand break repair. MATERIALS AND METHODS: XRCC4 with a HisTag were expressed by E. coli harboring plasmid DNA and purified. Phospho-mimicking mutants in which one phosphorylation site was replaced with aspartic acid were also prepared in order to reproduce the negative charge resulting from phosphorylation. The proteins were separated into dimers and multimers by gel filtration chromatography. Circular dichroism (CD) spectroscopy was performed in the region from ultraviolet to vacuum-ultraviolet. The CD spectra were analyzed with two analysis programs to evaluate the secondary structures of the wild-type and phospho-mimicked dimers and multimers. RESULT AND DISCUSSION: The proportion of ß-strand in the wild-type dimers was very low, particularly in their C-terminal region, including the five phosphorylation sites. The secondary structure of the phospho-mimic hardly changed in the dimeric form. In contrast, the ß-strand content increased and the α-helix content decreased upon multimerization of the wild-type protein. The structural change of multimers slightly depended on the phospho-mimic site. These results suggest that the ß-strand structure stabilizes the multimerization of XRCC4 and it is regulated by phosphorylation at the C-terminal site in living cells. CONCLUSION: An increase in the ß-strand content in XRCC4 is essential for stabilization of the multimeric form through C-terminal phosphorylation, allowing the formation of the large double-strand break repair machinery.

Incorporation of a bromine atom into DNA-related molecules changes their electronic properties.

To understand the mechanism underlying the high radio-sensitisation of living cells possessing brominated genomic DNA, X-ray photoelectron spectroscopy (XPS) using synchrotron X-rays with energies of 2000 or 2500 eV was used to study brominated and nonbrominated nucleobases, nucleosides and nucleotides. The bromine atom significantly reduced the energy gap between the valence and conduction states, although the core level states were not greatly affected. This finding was supported by quantum chemical calculation for the nucleobases and nucleosides. Our findings strongly indicate that the energy gaps between the valence and conduction levels of the molecules are significantly reduced by bromination. Furthermore, the brominated molecules are more likely to produce inelastic scattering low energy electrons upon exposure to 2000 or 3000 eV X-rays. This modification of electronic properties around the brominated group may both facilitate electron transfer to the brominated site in DNA and increase the probability of reaction with low energy electrons. These processes can induce DNA damage, presumably resulting in debromination of the uracil moiety and a subsequent cytotoxic effect.

Nature of the physicochemical process in water photolysis uncovered by a computer simulation.

In this work, we investigate the physicochemical process of water photolysis to bridge physical and chemical processes by a newly developed first-principles calculation code. The deceleration, thermalization, delocalization, and initial hydration of the extremely low-energy electrons ejected by water photolysis are sequentially tracked in the condensed phase. We show herein the calculated results for these sequential phenomena during 300 fs. Our results indicate that the mechanisms heavily depend on the intermolecular vibration and rotation modes peculiar to water and the momentum transfer between the electrons and the water medium. We suggest that using our results for the delocalized electron distribution will reproduce successive chemical reactions measured by photolysis experiments using a chemical reaction code. We expect our approach to become a powerful technique for various scientific fields related to water photolysis and radiolysis.

Initial yield of hydrated electron production from water radiolysis based on first-principles calculation.

Many scientific insights into water radiolysis have been applied for developing life science, including radiation-induced phenomena, such as DNA damage and mutation induction or carcinogenesis. However, the generation mechanism of free radicals due to radiolysis remains to be fully understood. Consequently, we have encountered a crucial problem in that the initial yields connecting radiation physics to chemistry must be parameterized. We have been challenged in the development of a simulation tool that can unravel the initial free radical yields, from physical interaction by radiation. The presented code enables the first-principles calculation of low energy secondary electrons resulting from the ionization, in which the secondary electron dynamics are simulated while considering dominant collision and polarization effects in water. In this study, using this code, we predicted the yield ratio between ionization and electronic excitation from a delocalization distribution of secondary electrons. The simulation result presented a theoretical initial yield of hydrated electrons. In radiation physics, the initial yield predicted from parameter analysis of radiolysis experiments in radiation chemistry was successfully reproduced. Our simulation code helps realize a reasonable spatiotemporal connection from radiation physics to chemistry, which would contribute to providing new scientific insights for precise understanding of underlying mechanisms of DNA damage induction.

Mitochondrial Metabolism in X-Irradiated Cells Undergoing Irreversible Cell-Cycle Arrest.

Irreversible cell-cycle-arrested cells not undergoing cell divisions have been thought to be metabolically less active because of the unnecessary consumption of energy for cell division. On the other hand, they might be actively involved in the tissue microenvironment through an inflammatory response. In this study, we examined the mitochondria-dependent metabolism in human cells irreversibly arrested in response to ionizing radiation to confirm this possibility. Human primary WI-38 fibroblast cells and the BJ-5ta fibroblast-like cell line were exposed to 20 Gy X-rays and cultured for up to 9 days after irradiation. The mitochondrial morphology and membrane potential were evaluated in the cells using the mitochondrial-specific fluorescent reagents MitoTracker Green (MTG) and 5,5',6,6'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1), respectively. The ratio of the mean MTG-stained total mitochondrial area per unit cell area decreased for up to 9 days after X-irradiation. The fraction of the high mitochondrial membrane potential area visualized by JC-1 staining reached its minimum 2 days after irradiation and then increased (particularly, WI-38 cells increased 1.8-fold the value of the control). Their chronological changes indicate that the mitochondrial volume in the irreversible cell-cycle-arrested cells showed significant increase concurrently with cellular volume expansion, indicating that the mitochondria-dependent energy metabolism was still active. These results indicate that the energy metabolism in X-ray-induced senescent-like cells is active compared to nonirradiated normal cells, even though they do not undergo cell divisions.

Analysis of differentially expressed genes on human X chromosome harboring large deletion induced by X-rays.

We examined here normal human cells with large deletions encompassing the hypoxanthine-phosphoribosyltransferase 1 (HPRT1) gene on X chromosome. Expression levels of genes on X chromosome were analyzed by microarray and RT-qPCR method, and differentially expressed genes (DEGs) were extracted. We found that DEGs were not limited to the genes flanking deleted regions but spread over the entire X chromosome. Interestingly, the gene regulation patterns were similar to a large extent among independent clones that have similar-sized large deletions involving the HPRT1 gene. Thus, it is indicated that an impact of large deletion on possible epigenetic transcriptional regulation is not limited to the regions proximal to the deletion region.

Secondary structural analyses of histone H2A-H2B proteins extracted from heated cells.

Histone proteins, building blocks of chromatins, participate in enzymatic reactions in cells heated at around 45°C though in vitro the denaturation of histones significantly proceeds at a similar temperature. It implies that unidentified mechanisms prevent thermal denaturation of histones in vivo. However, studies on the histone structures in the heated cells have been scarce. Here, we analyzed the secondary structures of histone H2A-H2B proteins originating from the heated cells using circular dichroism spectroscopy. The secondary structure contents of the H2A-H2B extracted from the heated cells differed from those of H2A-H2B both native and denatured in vitro but reverted to the native structures by incubating the heated cells at 37°C within 2 h. Such structural flexibility may play a role in protecting genomic functions governed by chromatin structures from heat stresses.

Electronic properties of DNA-related molecules containing a bromine atom.

PURPOSE: To clarify the radiosensitization mechanism masking the Auger effect of the cells possessing brominated DNA, the electronic properties of DNA-related molecules containing Br were investigated by X-ray spectroscopy and specific heat measurement. MATERIALS AND METHODS: X-ray absorption near-edge structure (XANES) and X-ray photoemission spectroscopy (XPS) were used to measure the electronic properties of the nucleotides with and without Br. We determined the specific heat of 5-bromouracil crystals with thymine as a reference molecule at low temperatures of 3-48 K to calculate the microscopic state numbers. RESULTS: Obtained XANES and XPS spectra indicated that both the lowest unoccupied molecular orbital (LUMO) and the core-levels were not affected by the Br incorporation. The state numbers of 5-bromouracil calculated from the specific heats obtained around 25 K was about 1.5 times larger than that for thymine below 20 K, although the numbers were almost the same below 5 K. DISCUSSION: Our results suggest that the Br atom may not contribute substantially to the LUMO and core-level electronic states of the molecule, but rather to the microscopic states related to the excitation of lattice vibrations, which may be involved in valence electronic states.

X-ray induced luminescence spectroscopy for DNA damaging intermediates aided by a monochromatic synchrotron radiation.

PURPOSE: To identify the bonding sites of initial radiation interaction with DNA and to trace the following chemical reaction sequences on the pathway of damage induction, we carry out a spectroscopy XIL (X-ray induced luminescence) using soft X-ray synchrotron radiation. This is a nondestructive analysis of the excited intermediate species produced in a molecular mechanism on the damage induction pathway. MATERIALS AND METHODS: We introduce aqueous samples of UMP (uridine-5'-monophosphate) in the vacuum by the use of a liquid micro-jet technique. The luminescence in the region of UV-VIS (from visible to ultraviolet) radiation induced after the absorption of monochromatic soft X-ray by aqueous UMP is measured with sweeping the soft X-ray energy in the region of 370-560 eV. RESULTS: The enhanced XIL intensities for aqueous UMP in the region of soft X-ray of 410-530 eV (in "water window" region) are obtained. The enhancement of XIL intensities in the UV-VIS region, relative to the water control, is explained by the excitation and ionization of a K-shell electron of nitrogen atoms in the uracil moiety. The enhanced XIL intensities do not match the structure of XANES (X-ray absorption near-edge structure) of the aqueous UMP. This suggests that the XIL intensities reflect the quantum yields of luminescence, or the quantum yields for conversion by UMP of an absorbed X-ray into UV-VIS radiation. In this paper, spectra of luminescence are shown to be resolved by combining low pass filters. The filtered luminescence spectra are obtained at the center of gravity (λc) of the band pass wavelength regions at λc = 270nm, 295 nm, 340 nm, 385 nm, 450 nm, and 525 nm., which show a trend similar to the fluorescence of nucleobases induced by ultraviolet radiation. CONCLUSION: It is concluded that the origin of the observed XIL is the hydrated uracil moiety in aqueous UMP, decomposition of which is suppressed by the migration of excess charge and internal energy after the double ionization due to Auger decay.

Core level ionization or excitation and Auger relaxation induce clustered DNA damage.

Ionizing radiation causes various types of DNA damage, such as single- (SSBs) and double-strand breaks (DSBs), nucleobase lesions, abasic sites (AP sites), and cross-linking between complementary strands of DNA or DNA and proteins. DSBs are among the most harmful type of DNA damage, inducing serious genetic effects such as cell lethality and mutation. Nucleobase lesions and AP sites, on the other hand, may be less deleterious and are promptly repaired by base excision repair (BER) pathways. Recently, biochemical approaches to quantify nucleobase lesions and AP sites have revealed certain types of non-strand break lesions as harmful DNA damage, called clustered DNA damage. Such clusters can retard nucleobase excision repair enzymes, and can sometimes be converted to DSBs by BER catalysis. This unique character of clustered DNA damage strongly depends on the spatial density of ionization or excitation events occurring at the track end of initial radiation or low energy secondary electrons. In particular, the photoelectric effect of elements comprising biological molecules, followed by emission of Auger electrons, are key factors in determining the future fate of each clustered damage site. This chapter describes biological studies of clustered nucleobase lesions with SSBs or AP sites, and mechanistical studies on core level excitation and Auger relaxation giving rise to clustered DNA damage.

Molecular Interactions of Normal and Irradiated Tubulins During Polymerization.

Microtubules, one of the cytoskeletons, are highly dynamic structures that play a variety of roles in maintaining cell morphology, cell division and intracellular transport. Microtubules are composed of heterodimers of α- and ß-tubulins, which are repeatedly polymerized and depolymerized. To investigate the radiation-induced impacts on the polymerization reaction of tubulins, we evaluated the molecular interactions between normal and irradiated tubulins. First, the polymerization reaction of the tubulins was measured after stepwise irradiation from 0 Gy to 1,000 Gy of X rays. The polymerization was inhibited in a dose-dependent manner. Next, the tubulins' polymerization reaction was then measured after the tubulin that was damaged from the exposure to 1,000 Gy of X rays was mixed with the normal tubulins. Our findings reveal that the radiation dose-dependent change in the degree of overall microtubule polymerization progression depends on the ratio of damaged tubulin. This result is biochemical evidence that non-DNA damage (in this case, cytoskeletal damage) from cytoplasmic radiation exposure may inhibit cell division, suggesting that some cytoskeletal damage may also affect the fate of the entire cell.

Establishment of a Method for Investigating Direct and Indirect Actions of Ionizing Radiation Using Scavenger-free Plasmid DNA.

In this study, an improved method using scavenger-free plasmid DNA was established to accurately determine yields of DNA damage induced by direct and indirect actions of ionizing radiation. The scavenger-free plasmid DNA was obtained by dialysis over 5-7 days, and the DNA solvent was replaced with phosphate buffer to completely remove impurities, which could be scavengers of radicals produced as a result of water radiolysis. DNA samples of films and dilute aqueous solutions were used to separately evaluate contributions of the direct and indirect actions of X rays (150-160 kVp). The irradiated DNA was analyzed by agarose gel electrophoresis to quantify strand-break yields. The yields of single-strand breaks (SSBs), n(SSB), were determined to be (6.5 ± 2.0) × 10-10 and (3.1 ± 0.9) × 10-7 SSBs/Gy/Da for the film and solution samples, respectively, showing a significant contribution of hydroxyl radicals (•OH) compared with direct energy depositions from ionizing radiation to DNA. As observed in SSBs, the yields of double-strand breaks (DSBs), n(DSB), were (5.6 ± 1.1) × 10-11 and (1.3 ± 0.2) × 10-8 DSBs/Gy/Da for the film and solution samples, respectively. The yield ratio of DSBs to SSBs, that is, n(DSB)/n(SSB), was 0.091 ± 0.026 for the film samples, while it was much lower for the solution samples (0.045 ± 0.010), indicating that direct actions result in more localized strand breaks relative to indirect actions. Base excision repair enzymes, namely, endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), were utilized after irradiations to convert base lesions and apurinic/apyrimidinic (AP) sites into strand breaks. The amounts of Nth and Fpg for the conversion were optimized to a few units per µg of DNA, although the optimal concentrations can differ among conditions.

A Brief Overview of Radiation-Induced Effects on Spermatogenesis and Oncofertility.

The genotoxicity of radiation on germ cells may be passed on to the next generation, thus its elucidation is not only a scientific issue but also an ethical, legal, and social issue in modern society. In this article, we briefly overview the effects of radiation on spermatogenesis and its associated genotoxicity, including the latest findings in the field of radiobiology. The potential role of transgenerational effects is still poorly understood, and further research in this area is desirable. Furthermore, from the perspective of oncofertility, we discuss the historical background and clinical importance of preserving male fertility during radiation treatment and the potential of microbeam radiotherapy. We hope that this review will contribute to stimulating further discussions and investigations for therapies for pediatric and adolescent/young adult patients.

No Intercellular Regulation of the Cell Cycle among Human Cervical Carcinoma HeLa Cells Expressing Fluorescent Ubiquitination-Based Cell-Cycle Indicators in Modulated Radiation Fields.

The non-targeted effects of radiation have been known to induce significant alternations in cell survival. Although the effects might govern the progression of tumor sites following advanced radiotherapy, the impacts on the intercellular control of the cell cycle following radiation exposure with a modified field, remain to be determined. Recently, a fluorescent ubiquitination-based cell-cycle indicator (FUCCI), which can visualize the cell-cycle phases with fluorescence microscopy in real time, was developed for biological cell research. In this study, we investigated the non-targeted effects on the regulation of the cell cycle of human cervical carcinoma (HeLa) cells with imperfect p53 function that express the FUCCI (HeLa-FUCCI cells). The possible effects on the cell-cycle phases via soluble factors were analyzed following exposure to different field configurations, which were delivered using a 150 kVp X-ray irradiator. In addition, using synchrotron-generated, 5.35 keV monochromatic X-ray microbeams, high-precision 200 µm-slit microbeam irradiation was performed to investigate the possible impacts on the cell-cycle phases via cell-cell contacts. Collectively, we could not detect the intercellular regulation of the cell cycle in HeLa-FUCCI cells, which suggested that the unregulated cell growth was a malignant tumor. Our findings indicated that there was no significant intercellular control system of the cell cycle in malignant tumors during or after radiotherapy, highlighting the differences between normal tissue and tumor characteristics.

Expression of an X-Ray Irradiated EGFP-Expressing Plasmid Transfected into Nonirradiated Human Cells.

To investigate the repairability of X-ray induced DNA damage, particularly non-double-strand breaks in living cells, enhanced green fluorescent protein (EGFP)-expressing plasmids X-ray irradiated and then transfected into nonirradiated human cells, MCF7 and MCF10A. Live-cell imaging of EGFP fluorescence was performed to measure the efficiency of plasmid repair in cells. The number of EGFP-expressing cells significantly decreased with increasing X-ray dose for both cell lines. The obtained kinetic curves of EGFP expression indicating plasmid repair were quantitatively compared against algebraically calculated ones based on the values of the transfected plasmids that had been treated with nicking or restriction enzymes. Then, assuming a Poisson distribution of single-strand breaks (SSBs), the number of cells carrying these nicked plasmids that could express EGFP were estimated. Our experimental results revealed considerably fewer cells expressing EGFP compared to the expected values we had calculated. These results suggest that the lower proportion of cells expressing EGFP as a measure of plasmid repair was due not only to the complex chemical structures of termini created by SSBs compared to those created by enzyme treatments, but also that base lesions or AP sites proximately arising at the strand-break termini might compromise EGFP expression. These results emphasize that radiation-induced DNA breaks are less repairable than enzymatically induced DNA breaks, which is not apparent when using conventional gel electrophoresis assays of plasmid DNA.

Molecular Configuration of Human Genome Neighboring Megabase-Sized Large Deletions Induced by X-Ray Irradiation.

The genomic landscape neighboring large deletions including the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on human X chromosome in 6-thioguanine-resistant mutants originating from immortalized human fibroblast cells exposed to X rays was characterized by real-time quantitative PCR (qPCR)-based analyses. Among the 13 mutant clones with large deletions extending over several Mb, including the HPRT locus, revealed by 10 conventional sequence-tagged site (STS) markers, three clones bearing the largest deletions were selected for further qPCR analysis using another 21 STS markers and 15 newly designed PCR primer pairs. The results indicated that the major deletions were in very specific regions between the 130-Mb and 140-Mb positions containing the HPRT locus on the X chromosome and, contrary to our initial expectations, additional minor deletions were distributed in a patchwork pattern. These findings strongly indicate that the complex deletion patterns in the affected chromosome are related to the radiation track structure with spatially heterogeneous energy deposition and the specific structure of the chromatin-nuclear membrane complex. The uncovered complex deletion patterns are in agreement with the idea of complex chromatin damage, which is frequently associated with carcinogenesis.