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To understand the mechanism underlying the high radio-sensitisation of living cells possessing brominated genomic DNA, X-ray photoelectron spectroscopy was used to study brominated and nonbrominated nucleobases, nucleosides and nucleotides.

For water radiolysis, conventional simulation methods estimate free radical yields based on the cross-sections. Our results indicated that electron dynamic motion must be further solved to predict the initial yields of hydrated electrons.

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.

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.

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.

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.

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.

Tissue is a complex system with a highly dynamic structure to maintain a stable internal environment. The homeostatic mechanisms could be involved in radiation-induced effects at the tissue level. Also, tissue-weighting factors have been regarded as an essential index for the guideline released from the International Committee on Radiological Protection. Thus, for both radiobiology and health physics, it is important to understand radiation-induced tissue-level responses. The applications of microbeams and tissue cultures illustrated in this article focus on their relevance to radiation tissue biology that provides a novel approach for greater accuracy in risk assessment of radiation exposure. We review on recent progress in both microbeams and tissue cultures, which is promising for providing novel insights into radiation tissue biology. In addition, we present the use of ex vivo mouse testicular tissue culture as an experimental model of spermatogenesis to investigate radiobiological effects with microbeams. Our model represents a unique application in the field, with significant potential for deeper mechanistic insight into radiation-induced tissue-level effects and appropriate radiological protection.

Spatially fractionated radiation therapy (SFRT) has been based on the delivery of a single high-dose fraction to a large treatment area that has been divided into several smaller fields, reducing the overall toxicity and adverse effects. Complementary microbeam studies have also shown an effective tissue-sparing effect (TSE) in various tissue types and species after spatially fractionated irradiation at the microscale level; however, the underlying biological mechanism remains elusive. In the current study, using the combination of an ex vivo mouse spermatogenesis model and high-precision X-ray microbeams, we revealed the significant TSE for maintaining spermatogenesis after spatially fractionated microbeam irradiation. We used the following ratios of the irradiated to nonirradiated areas: 50:50, 150:50 and 350:50 lm-slit, where approximately 50, 75 and 87.5% of the sample was irradiated (using center-tocenter distances of 100, 200 and 400 lm, respectively). We found that the 50 and 75% micro-slit irradiated testicular tissues showed an almost unadulterated TSE for spermatogenesis, whereas the 87.5% micro-slit irradiated tissues showed an incomplete TSE. This suggests that the TSE efficiency for spermatogenesis is dependent on the size of the nonirradiated spermatogonial stem cell pool in the irradiated testicular tissues. In addition, there would be a spatiotemporal limitation of stem cell migration/competition, resulting in the insufficient TSE for 87.5% micro-slit irradiated tissues. These stem cell characteristics are essential for the accurate prediction of tissue-level responses during or after SFRT, indicating the clinical potential for achieving better outcomes while preventing adverse effects.

XRCC4 is a key protein to repair severe DNA damage of double strand breaks (1). The XRCC4 dimer has been reported to form a multimer scaffold with another repair protein, the XRCC4-like factor (XLF). Enzymatically induced phosphorylation at several amino acid residues in XRCC4, such as serine (2), might cause a local change of statically electric charge, resulting conformational alteration to activate the protein. However, the correlation between the structural change and activation has not been understood yet. Full-length XRCC4 was not able to be crystallized because of its intrinsically disordered C-terminal region including several phosphorylation sites. Previously, we have applied circular dichroism (CD) spectral analysis for the full length of wild type XRCC4 (denoted as WT) in an aqueous solution and reported its characteristic secondary structure (3). In the present study, we focused further the structural change by phosphorylation. We prepared a dimer and multimers of WT XRCC4 as well as mutated ones in which serine residues were substituted to an aspartic acid (denoted as S260D and S327D) to mimic phosphorylation in terms of statically electric charge. Spectra of XRCC4 dimer proteins were similar irrespective of phosphorylation. On the other hand, spectra of multimers were significantly different between WT and mutated proteins (FIGURE 1). The composition ratios of the secondary structures were calculated for each sample using a software, SELCON3 (TABLE 1). The results indicate that the multimerization and pseudo-phosphorylation increase β-strands. Small angle X-ray scattering (SAXS) at BL6A at the Photon Factory in KEK was also applied to further obtain structural information of XRCC4. The results suggest that the XRCC4S260D dimer has a slightly larger radius of inertia (Rg) than WT (TABLE 2), which may correspond to the slight increase in β-strands shown in the CD analysis. The β-strand formation by the phosphorylation at the C-terminal might stabilize the NHEJ machinery after DSB repair completion., The 26th Hiroshima International Symposium on Synchrotron Radiation

ケージド化合物は生物活性を持つ分子に光分解性の保護基を結合させることで不活性化した化合物であり、保護基の特定の共有結合を光化学的に切断することにより再活性化できる。ケージド化合物は光照射により空間的・時間的に生物活性を操作できることから生体内の狙った場所での薬剤放出等への応用が期待されている。ケージド化合物の活性化には可視から近赤外領域の光が使用されているが、透過性が低いため生体深部への適用は困難である。そこで、生体透過性の高いX線と特異的に反応する保護基を用いたケージド化合物の開発を目指している。X線照射により活性化できるケージド抗がん剤に用いる保護基の候補として、高エネルギーX線に対する吸収断面積が大きく、選択的な結合切断を誘導することが期待される超原子価ヨウ素化合物に着目した。本研究では、超原子価ヨウ素化合物である2-ヨードソ安息香酸(IBA)とジアセトキシヨードベンゼン(PIDA)のＸ線に対する反応性を評価するため、ヨウ素L3殻吸収端近傍のエネルギー領域でXANESスペクトルを測定した。 XANESスペクトルは、KEK-PFのBL-27Bで試料粉末をScotchメンディングテープに塗布して透過法により測定した。 IBAと2-ヨード安息香酸は特徴的な4つの、またPIDAは5つの吸収ピークを示した。2-ヨード安息香酸と比較すると超原子価ヨウ素化合物の共鳴吸収の強度は大きく、そのピークは高エネルギー側にシフトした。当日は、吸収ピークに対応する単色X線照射によって生じる脱離種に関しても議論する予定である。, 021年度量子ビームサイエンスフェスタ 第13回MLFシンポジウム, 第39回PFシンポジウム

DNA中のチミンを5-臭化ウラシル（BrU）に置換すると、放射線に対する致死率が増加する（放射線増感作用）ことが知られているが、その物理化学的なメカニズムは明らかになっていない。最近、平戸らはチミンとBrUのX線光電子分光（XPS）測定を行い、光電子強度が0になる結合エネルギーE0がチミン1.8 eVに対してBrUではほぼ0 eVになることを示した。このような電子構造の差が放射線増感作用の起源となっている可能性がある。そこで本研究では、他のハロゲン化ウラシル、5-フッ化ウラシル（FU）、5-塩化ウラシル(ClU)、5-ヨウ化ウラシル（IU）、およびウラシル（U）のXPS測定を行い、その電子状態を調査したので報告する。 U、FU、ClU、IUのペレット（直径10 mm、厚さ0.3 mm）を試料として用いた。XPS測定はPF BL-27Aで行った。励起光のエネルギーは2 keVとした。 UのE0が2.2 eVであったのに対し、FU、ClU、IUのそれはそれぞれ1.4、0.8、0.2 eVとなり、ハロゲン化あるいはハロゲンの原子番号が大きくなるほどスペクトルの立ち上がりが低エネルギー側にシフトした。臭素の原子番号はヨウ素よりも小さいが、BrUのE0はIUよりも低く、この傾向から外れていた。IUも放射線増感作用を示すことから、価電子帯の電子構造、特にE0の大きさ、が放射線増感作用の大小に関わっている可能性があると結論した。, 2021年度量子ビームサイエンスフェスタ

Several studies have demonstrated that mitochondria are critically involved in the pleiotropic manifestation of radiation effects. While conventional whole-cell irradiation compromises the function of mitochondria, the effects of subcellular targeted irradiation are not yet fully understood. In this study, normal human diploid cells with cell-cycle indicators were exposed to a synchrotron X-ray microbeam, and mitochondrial membrane potential was quantified by JC-1 over the 72-h period after irradiation. Cytoplasmic irradiation was observed to temporarily enlarge the mitochondrial area with high membrane potential, while the total mitochondrial area did not change significantly. Unexpectedly, cell-nucleus irradiation promoted a similar increase not only in the mitochondrial areas with high membrane potential, but also in those with low membrane potential, which gave rise to the apparent increase in the total mitochondrial area. Augmentation of the mitochondrial area with low membrane potential was predominantly observed among G1 cells, suggesting that nucleus irradiation during the G1 phase regulated the mitochondrial dynamics of the cytoplasm, presumably through DNA damage in the nucleus

Simple Summary Spermatogenesis is one of the most important processes for the propagation of life; however, the testes' ability to form sperm via this differentiation process is highly radiosensitive and easily impacted by exposure to environmental, occupational, or therapeutic radiation. Furthermore, the possibility that radiation effects on the gonads can be passed on from generation to generation should not be overlooked. This review focuses on the radiation-induced effects on spermatogenesis and the transgenerational effects. We also explore the potential of novel radiobiological approaches to improve male fertility preservation during radiotherapy. 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.

RNA塩基であるウラシルの5位の炭素をハロゲン化した5-ハロウラシル（図1左: XU; X = F, Cl, Br, I）の窒素、酸素のK殻励起に伴う不対電子生成量は、高い放射線増感作用を示すBrUやIUで少なく、増感能の低いClUで多くなることが知られている。我々のグループは、これらの性質がハロゲン近傍の電子状態に起因すると考え、その検証を行っている。本研究では、ClUの塩素K殻吸収端XANESスペクトル測定を行ったので報告する。 試料には、ClUの粉末、水溶液を用いた。XANESスペクトルはPF BL-27Aにおいて蛍光収量法により測定した。ClUがDNAに取り込まれた場合を想定し、ClUにデオキシリボースが結合したCldU（図1右）についても同様の測定を行った。また、DV-Xα計算によりピークの帰属を行った。 粉末、水溶液どちらの場合でも、CldUの第一ピーク（Cl 1s→σ*）の吸収強度がClUのそれよりも大きくなり、塩素を含まないデオキシリボースの付加が塩素K殻の電子励起に影響を与えることが示された。当日は、XANESスペクトルおよび理論計算結果の詳細について議論する予定である。, 第35回日本放射光学会年会・放射光科学合同シンポジウム

Photoimmuno-therapy (PIT) has been recognized as one of the most promising methods in advanced cancer therapies. The technique applies photosensitizing molecules, particularly phthalocyanine (IR700), conjugated to monoclonal antibodies (mAbs) targeting epidermal growth factor receptors (EGFR) of cancer cell membranes. After irradiating the mAb-IR700-bound target cells with near-infrared (NIR) light, the axial ligands are released from the photosensitizers and, thereby, the cell membranes become brittle resulting prompt cell death. Although the evidences clearly show a strong potential of PIT, the practical penetration depth of NIR is less than a few cm and this might be controversial when it is applied to other deeply located tumors. To overcome this, we have examined X-ray induced core level excitation as alternative initiation probes. X-rays further penetrate in tissues than NIR. In the present study, the reaction probability of phthalocyanine with X-rays were evaluated by measuring atom selective K-shell X-ray absorption/excitation spectrum of IR700 using a synchrotron soft X-ray beamline. The cross sections of nitrogen (N), oxygen (O), and silicon (Si) atoms were evaluated using X-ray absorption fine structure (XANES) spectra, and reveled that O K-shell resonance excitation showed a larger cross section than those for N or Si, 2 hold of that at just below K-edge energy. This could be effective to detach the axial ligands because the oxygen atoms link the ligands and molecular body. Monte Carlo track simulation was applied to estimate the range of photo- and Auger electrons emitted by the K-excitation/ionization.Low energy Auger electrons showed noticeably short ranges less than a few ten nanometer and gave their energy within the molecular size of phthalocyanine. These results indicate that these secondary electrons may also contribute to the ligand release., Pacifichem 2021

小胞体は、最大の細胞小器官で、粗面小胞体と割面小胞体に分かれる。粗面小胞体は、タンパク質の合成と折りたたみ、滑面小胞体は脂質の合成、カルシウムイオンの貯蔵といった役割をもつ。本研究では、放射線照射後の小胞体に与える影響について、形態的な変化に着目して明らかにすることを目的とし、これの観察方法の確立を目指した。まず、小胞体染色試薬での染色時間について、検討を行った。染色時間を15分、20分30分と変えて観察開始時点での蛍光強度を比較した結果、染色時間20分での蛍光強度が最も高く、染色時間は20分が最適であった。次に、X線を1Gy/minの線量率で6 Gy照射し、小胞体を24時間観察した。その結果、24時間小胞体を観察することが可能であると判断された。また、細胞周囲に気泡状の構造がコントロールとX線照射サンプルの両方の細胞で観察され、その細胞数はX線照射サンプルで有意に多かった。この構造は、放射線照射によって生じた異常なタンパク質が小胞体に蓄積した結果現れたものではないかと考えている。, 量子生命科学先端フォーラム2021年冬の研究会

本研究では、線形化されたDNAがリニアの状態で細胞内にトランスフェクトされ、そのDNAの切断部位とEGFP遺伝子までの距離に応じてトランスフェクション効率が変化するかどうかを調査した。結果として、コントロールと比較すると酵素処理のDNAのトランスフェクション効率が低くなった。しかし、EGFP遺伝子がリニアDNAの中央付近に位置しているStu１処理したサンプルは、EGFP遺伝子が末端に位置している他の酵素処理サンプルより2倍近いトランスフェクション効率を示した。これらのことから線形化されたDNAは、蛍光遺伝子とDSB末端の相対的な位置に依存して、ある程度細胞にトランスフェクトされ、蛍光タンパク質を発現する能力を持っている可能性が示唆された。, 量子生命科学先端フォーラム 2021冬の研究会

放射線がDNAなどの生体分子と衝突すると、エネルギーの低い二次電子が生じる場合があり、一時的に分子がアニオンになった後に解離することがある。これを解離性電子付着という。未だに解離性電子付着によるDNA損傷やその修復メカニズムについて明らかになっていないことが多く、早急な解明が求められている。本研究では、低エネルギーX線照射によって生成されたDNA損傷の収率と、非照射細胞内における修復難易度を調べることを目的とした。これを、アガロースゲル電気泳動と生細胞タイムラプス撮影の二点で検討を行った。プラスミドDNAは損傷によって、コンフォメーションが変化する。そのDNAの立体構造の変化を、アガロースゲル電気泳動により分析し、1/e線量を算出した。そして、1/e線量を照射したDNAプラスミドDNAサンプルを、トランスフェクションによって非照射細胞に導入し、EGFP発現をライブセルにてタイムラプス撮影し、経時的に観察を行った。今回は、これらの実験結果について発表する。, 量子生命科学先端フォーラム2021冬の研究会

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

Radicals produced in crystalline L-alanine-3,3,3-d3 and L-alanine-d4 were observed by the electron spin reso-nance (ESR) technique during 1.5 keV soft X-ray irradiation. The spectral change from •CHCD3COOH to •CDCD3COOH by a hydrogen exchange reaction was directly observed in L-alanine-3,3,3-d3. The line width of the ESR spectra obtained by the soft X-ray irradiation was 1.5 times wider than that of hard X-rays from a previous report, meaning a higher density of radicals.

We have established an experimental procedure of transfection and expression of irradiated plasmid DNA encoding fluorescent protein, EGFP, in eukaryotic cells. In this assay, the number of cells expressing the fluorescent protein increases with the incubation of cells as DNA damage is repaired. When a DSB-induced linear plasmid DNA arises after irradiation, it has been thought that the plasmid could be enzymatically digested immediately after transfection or delivery into nucleus. Previously we found that the linear plasmid DNA obtained by restriction enzyme treatments shows slightly expresses EGFP [1]. However, it has not yet been clarified how the linear DNA expresses after transfection. In this study, we aim to elucidate the efficiency of the linear plasmid DNA expression of EGFP in human cells. The linear plasmids were prepared by treatments with various restriction enzymes with different recognition sequences. We cleaved the plasmid around the EGFP gene with three blunt end restriction enzymes at the upstream side, the downstream side, and a 1.2 kbp distance away from the downstream sides of the EGFP gene. Preliminary results indicate that EGFP-expressing was observed for the linear plasmid transfections. The farther the cleavage site from the EGFP gene induced the higher transfection efficiency, suggesting that the plasmids might progressively be digested from the unstable terminal ends when they are transfected into cells. [1] Obata et al. Rad. Res. (2021) In press., 日本放射線影響学会第64回大会

Cells with DNA incorporating Br-dUMP as a thymidine monophosphate (dTMP) analog are known to be highly sensitive to ionizing radiation. To clarify physicochemical mechanism of the bromine-radiosensitization, the electronic state of the Br-DNA related molecules was investigated applying X-ray photoelectron spectroscopy (XPS). Quantum chemical calculations were also performed for these molecules which were undergone structural optimization using the Hartree-Fock (HF) and Møller-Plesset (MP2) method, and Density Functional Theory (DFT) to investigate whether the bromination affects the highest occupied and lowest unoccupied orbital (HOMO and LUMO) of these molecules. In terms of “radiation effect”, total yield of low-energy electrons generated by inelastic scattering of photo- or Auger electrons were also quantified using the XPS apparatus. The main findings from this study on the bromination of DNA are the following: (i) little effect on the core levels, the LUMO shapes and their spatial distributions for all atoms, (ii) contrarily reducing the binding energy of valence electrons to 0 eV from 1.0-1.8 eV, (iii) a high efficiency in generating the low energy electrons. These results suggest that the presence of Br reduce an energy gap between HOMO and LUMO so that it facilitates transfer of charges, holes, or the low energy electrons. The brominated DNA would possess a half-metal-like electronic property localized (presumably a nucleotide level) around bromine atom leading finally deleterious DNA damage in cells., 日本放射線影響学会第64回大会

Recent studies reported that, in oncogene induced senescence, autophagy is activated during senescence initiation period to ensure rapid protein synthesis. However, senescent cells undergo metabolic and epigenetic changes during their long survival period, and autophagic activity might fluctuate with these changes. To elucidate whether autophagy fluctuates through the progress of senescence, we investigated temporal activity of autophagy over 30 days in normal human diploid cells, hTERT BJ-5ta, exposed to 20 Gy X-rays. Based on the expression levels of p21 and p16, we confirmed that the senescence-like growth arrest was surely induced after irradiation, and senescence was completely established by 7 days post-irradiation. We measured autophagic activity using quantification of LC3-II which is localized in autophagosomes. Autophagosomes are degraded by fusing with lysosomes. Autophagic activity consequently needs to be evaluated including the amount of degraded autophagosomes. We used chloroquine (CQ) as an inhibitor of lysosomal degradation and determined net LC3-II levels including those in the degraded autophagosomes. We found that LC3-II level increased to the same level as control under CQ treatment condition at 1 day after irradiation. At 3 and 7 days, the LC3-II levels with CQ were significantly lower compared to that of control with CQ. At 30 days, it turned to elevate. These results indicate that, in X-ray irradiated cells, autophagy is not significantly activated during senescence initiation, but activated after senescence is established. This delayed activation of autophagy is not seen in oncogene induced senescence, thus it might be due to radiation damage to organelles in cytoplasm., 日本放射線影響学会第64回大会

It is becoming recognized that cellular temperature depends on the intracellular condition. The heat balance of chemical reactions inside various organelles may change significantly due to external stress, such as ionizing irradiation. In the present study, HeLa cells were investigated a fluorescent chemical probe, Cellular Thermoprobe for Fluorescence Ratio, to determine temperature of each cell after X-ray irradiation of 10 Gy. We used two methods to irradiate the cells with X-rays. The first is to irradiate the entire dish with X-rays. The second method is to limit the irradiation range and irradiate X-rays separately in the irradiated area and the non-irradiated area. After irradiation with X-rays, it was observed by the microscope. The intensity and distribution of the two fluorescences were analyzed using an image analysis software, ImageJ. The temperatures were then determined from the obtained fluorescent ratios based on a calibration curve. The intracellular temperature were significantly different even between cells in same dish. For the irradiated cells, the temperature was more dispersed among the cells and prone to be statically higher than that of the control cells. Fine temperature-distribution over the cell including various organelles will be also reported., 日本放射線影響学会第64回大会

We have established an experimental procedure of transfection and expression of irradiated plasmid DNA encoding fluorescent protein, EGFP, in eukaryotic cells. In this assay, the number of cells expressing the fluorescent protein increases with the incubation of cells as DNA damage is repaired. When a DSB-induced linear plasmid DNA arises after irradiation, it has been thought that the plasmid could be enzymatically digested immediately after transfection or delivery into nucleus. Previously we found that the linear plasmid DNA obtained by restriction enzyme treatments shows slightly expresses EGFP [1]. However, it has not yet been clarified how the linear DNA expresses after transfection. In this study, we aim to elucidate the efficiency of the linear plasmid DNA expression of EGFP in human cells. The linear plasmids were prepared by treatments with various restriction enzymes with different recognition sequences. We cleaved the plasmid around the EGFP gene with three blunt end restriction enzymes at the upstream side, the downstream side, and a 1.2 kbp distance away from the downstream sides of the EGFP gene. Preliminary results indicate that EGFP-expressing was observed for the linear plasmid transfections. The farther the cleavage site from the EGFP gene induced the higher transfection efficiency, suggesting that the plasmids might progressively be digested from the unstable terminal ends when they are transfected into cells. [1] Obata et al. Rad. Res. (2021) In press., 日本放射線影響学会第64回大会

捕捉剤フリーの条件においてX線が誘発するDNA損傷の収率を調べた。DNA試料の含水率を変えることで直接作用と間接作用の寄与を制御した。DNA損傷は一本鎖切断（SSB）、二本鎖切断（DSB）、及び塩基損傷（base lesion）といったタイプごとに定量した。いずれのDNA試料に生じた複雑な損傷も、直接作用によって誘発されるものと示唆された。, 第 64 回放射線化学討論会

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 non-destructive 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 eV – 560 eV. Results: The enhanced XIL intensities for aqueous UMP in the region of soft X-ray of 410 eV – 530 eV (in “water window” region) are obtained (Kojima et al, 2019). 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 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, 340nm, 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.

外部環境からのストレスを受けた多細胞生物の細胞は、周囲の細胞や細胞外液からのシグナルを伝搬・受容する仕組みを備えている。例えば、細胞にあらかじめ低線量放射線を照射した後、高線量の放射線を照射すると、事前照射を行わなかった場合と比較して放射線影響が緩和されることが知られており、一般に放射線適応応答と呼ばれている。放射線適応応答では、IP3やCaMが関与する複雑なCa2+放出反応ネットワークが形成されており、脳・神経細胞で細胞内カルシウムストアである小胞体によるフィードバック経路を介した時空間的な振動構造が知られている。 情報伝達物質の周期的な放出と受容による信号伝達を行う細胞集団は、外力に対して頑強性（ロバストネス）を持つ非線形振動子として振る舞うと推測される。外部刺激を受けた細胞集団におけるシグナル伝達物質の時空間的な濃度変化（疎密波）を、生細胞のタイムラプス撮影（in vivo）と生態学数理モデルを応用した解析（in silico）の二点から検討した。 Ca2+濃度変化のライブセルイメージング法の検討として、ヒト正常繊維芽細胞（BJ-1 h-TERT）に対してCa2+に特異的な化学的蛍光プローブ（Fluo 4-AM）を導入し、蛍光顕微鏡を用いて細胞全体の輝度の時系列変化を約5分間撮影した（下図）。タイムラプス撮影の結果から、細胞内の相対的なCa2+濃度変化を算出した。数理モデル解析では、細胞内のCa2+波におけるCaM、IP3R、IP3によるネガティブフィードバックに着目し、生態学における生物の捕食－被食関係を表す数理モデルとして知られるロトカ・ヴォルテラ非線形連立微分方程式をCa2+濃度振動の解析に応用した。本実験ではまずCa2+濃度振動による情報伝達ネットワークを簡略化し、小胞体のCa2+放出に伴うCaM濃度の減少と、外部刺激によるCa2+の一過性上昇を考慮したモデルを作成した。 ライブセル観察の結果より、細胞に観察時の励起光の照射効果が見られた他、隣接する細胞群の中には、Ca2+濃度変化のピーク強度や半値幅に細胞間の距離との相関があることが推測でき、これらの値が情報伝達の閾値として機能している可能性があると考えられる。情報伝達の起点となる細胞からの距離に比例/反比例して疎密波の波形が変化すると推測される。また、ロトカ・ヴォルテラ方程式を基盤とした細胞内Ca2+濃度振動のモデル化により、Ca2+濃度振動の定性的な再現を行うことができた。 今後は細胞にX線を照射した場合の細胞内Ca2+濃度変化を算出し、非照射群との比較・検討を行う。また、マイクロビームで細胞の部分照射を行い、細胞内外のCa疎密波による情報伝達過程の理解を目指す。数理モデル解析については、現状のモデルを改良することでより複雑な細胞内カルシウム動態のモデルを構築し、さらには細胞間のCaシグナル伝達に関するモデルを構築することで、ライブセル実験によって得られた結果との比較検討が将来期待できる。, 第58回アイソトープ・放射線研究発表会

放射線誘発DNA損傷における直接作用と間接作用の寄与は、DNA試料の含水率で決まる。含水率を制御してX線照射を行い、鎖切断や塩基損傷などのDNA損傷の収率を評価した。DNA損傷の検出にはアガロースゲル電気泳動法を用いた。微量薬剤の添加による影響の検討を視野に入れ、捕捉剤フリーの高純度DNA試料を用いた。捕捉剤を含む試料での報告との比較から、捕捉剤ごとにDNA損傷を選択的に防護することが示された。, 第58回アイソトープ・放射線研究会発表会

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.

Precision medicine is a rapidly developing area that aims to deliver targeted therapies based on individual patient characteristics. However, current radiation treatment is not yet personalized; consequently, there is a critical need for specific patient characteristics of both tumor and normal tissues to be fully incorporated into dose prescription. Furthermore, current risk assessment following environmental, occupational, or accidental exposures to radiation is based on population effects, and does not account for individual diversity underpinning radiosensitivity. The lack of personalized approaches in both radiotherapy and radiation risk assessment resulted in the current situation where a population-based model, effective dose, is being used. In this review article, to stimulate scientific discussion for precision medicine in both radiotherapy and radiation risk assessment, we propose a novel radiological concept and metric – the personalized dose and the personalized risk index – that incorporate individual physiological, lifestylerelated and genomic variations and radiosensitivity, outlining the potential clinical application for precision medicine. We also review on recent progress in both genomics and biobanking research, which is promising for providing novel insights into individual radiosensitivity, and for creating a novel conceptual framework of precision radiotherapy and radiation risk assessment

We applied circular dichroism (CD) spectral analysis to obtain the secondary structure of the DNA repair protein XRCC4, with a focus on its C-terminus. Using synchrotron radiation as a light source across a wide range of wavelengths, including vacuum ultraviolet (UV) light from 180 to 200 nm and UV light from 200 to 240 nm, we determined the secondary structure composition of the full-length protein, including its C-terminus, which had not yet been -the focus of crystallography studies, though it contains several phosphorylation sites. The secondary structures inferred using the obtained CD spectra indicate that the C-terminus is composed of a substantial fraction of turns with a few unordered and alpha-helix structures and almost no beta-strands. The C-terminus is likely to form a characteristic secondary structure with turns as a main component, and its DNA repair function is likely regulated by the structural change induced by phosphorylation of the terminus.

To investigate the repair process of DNA damage induced by ionizing radiation in isolation from various types of cytoplasmic damage, we transfected X-irradiated enhanced green fluorescent protein (EGFP)-expressing plasmid DNA into non-irradiated mammalian cells using lipofectamine. The repair kinetics of the irradiated plasmids in the cells were visualized under microscopy as the EGFP fluorescence emitted by transfected cells. Using an agarose gel electrophoresis method, the yields of single- and double-strand breaks of the plasmids were also quantified. As positive control experiments, plasmid DNA with single- or double-strand breaks induced by a nicking or restriction enzyme were also transfected into the cells. The DNA repair rates for X-ray-irradiated plasmids were significantly lower than those of the enzymatically digested positive control samples. These results indicate that X-rays could induce less repairable damage than that induced by enzymes.

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 β-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.

放射線に誘発されるDNA 変異の起点は、直接作用と間接作用に分類される。後者の過程に着目し、捕捉剤フリーな条件でX 線照射した際に生成される塩基損傷の収率を調べた。また、TE 緩衝液に含まれる溶質の防護効果についても検討した。プリン塩基損傷の方がピリミジン塩基より高い収率になる傾向あり，過去のTE 緩衝液で行われた研究とは逆の傾向を示した。このことからTE 緩衝液はプリン塩基損傷を選択的に防護すると推測され、抗酸化剤の種類によって防護の選択性に違いが見られる可能性が示唆された。