Microscopic imaging of DNA repair foci in irradiated normal tissues

Purpose:?It is now feasible to detect DNA double strand breaks (DSB) in tissues by measuring the induction and resolution of DNA repair foci, such as γ-H2AX, using immunofluorescent microscopy and digital image analysis. This review will highlight principal tools and approaches to tissue microscopy and analysis. It will also discuss the practical considerations of using microscopy in vitro and in vivo in measuring intranuclear foci following irradiation.

Conclusions:?Computer-based image analysis algorithms allow an objective and quantitative analysis of foci and protein-protein interactions using 3D confocal images. Finally, we review the literature in which DNA repair foci have been investigated as a biodosimeter or a biomarker of DNA repair in normal tissues.     

Variability in the Oxygen Effect Observed with Micro-organisms

Post-irradiation conditions of culture are considerably more effective in altering the response of E. coli B to ionizing radiation if this has been delivered in anoxic conditions. Thus the oxygen-enhancement ratio depends on conditions of culture, and has been found to vary from 1·6 to 3·7. A linear relationship exists between 37 per cent dose and oxygen-enhancement ratio. This supports the inference that radiation causes lethal damage by inflicting at least two different types of injury, one of which is much more affected by the presence of oxygen during irradiation.     

丁烯酸内酯对大鼠的脂质过氧化效应

目的：研究了丁烯酸内酯的脂质过氧化效应。方法：采用硫代巴比妥酸（ＴＢＡ）显色法及巯基含量测定等方法测定了脂质过氧化的代谢产物。     

T－2毒素对肝脏脂质过氧化作用的体外效应

在体外观察了Ｔ－２毒素对大鼠及小鼠肝脏脂质过氧化的影响。结果表明，１－２毒素引起肝匀浆脂质过大化物丙二醛及脂褐素含量降低，并且这种变化存在时间和剂量效应关系；不同剂量Ｔ－２毒素对肝匀浆总巯基、蛋白巯基以及非蛋白巯基无明显影响。     

Dose-length product of scanners correlates with DNA damage in patients undergoing contrast CT

Objectives

Computed tomography (CT) exams contribute for a large part to the population's radiation burden. This study addresses the question if dose settings of scanners expressed by dose-length product (DLP) are correlated with directly measurable biological effects in patients.

Methods

DLP, blood dose, effective dose and DNA damage were analyzed for patients undergoing a thoracic or abdominal contrast CT scan on two CT scanners with different dose settings. The DNA damage was assessed by scoring γ-H2AX foci representing DNA double-strand breaks (DSBs) in patient's lymphocytes. Blood dose was calculated using the ImPACT software.

Results

The CT system operating at higher dose settings represented by higher DLP values, resulted in a significantly higher number of radiation-induced γ-H2AX foci in patient's lymphocytes (DLP: 2.1 times higher; γ-H2AX foci: 2.3 times higher; p < 0.05). Plotting γ-H2AX foci versus blood dose showed a systematic increase of DNA damage with dose. In vitro experiments ruled out a possible X-ray enhancement of DNA damage effect by contrast agent.

Conclusions

Present study demonstrates that optimization of DLP setting of scanners results in a reduction of X-ray effects in patients.     

香兰素衍生物VND3207对小鼠骨髓细胞遗传损伤的防护效应研究

目的 研究香兰素衍生物4-羟基-3,5-二甲氧基苯甲醛(VND3207)对γ射线整体照射小鼠骨髓细胞遗传损伤的防护作用。方法 BALB/c小鼠按10、50和100 mg/kg不同剂量灌胃给药,1次/d,连续5 d,最后一次给药后2 h, 2 Gy ⁶⁰Co γ射线照射,照射后48 h观察小鼠骨髓嗜多染红细胞微核、染色体畸变和骨髓有丝分裂指数的改变。结果 不同剂量(10、50和100mg/kg)的VND3207均能有效地降低小鼠骨髓嗜多染红细胞微核率(t=2.40～4.26,P<0.05)和染色体畸变率(t=2.36～3.52,P<0.05),同时提高骨髓有丝分裂指数。药物保护效果与给药剂量呈依赖关系,其中100 mg/kg剂量组的微核率和染色体畸变率与单纯照射组相比,分别降低了65%和50%。结论 VND3207对⁶⁰Co γ射线诱发的小鼠骨髓辐射损伤有良好的保护作用。     

Comparison of methods to quantify histone H2AX phosphorylation and its usefulness for prediction of radiosensitivity

Abstract

Purpose: The number of radio-induced double-strand breaks is correlated with the number of histone gamma-H2AX (γ-H2AX) foci. For this reason, foci quantification is a useful tool to measure radiation-induced DNA damage and the number of foci has been suggested as a predictive biomarker of radiosensitivity. The aim of the present study was to evaluate the reproducibility of different microscopic methodologies and flow cytometry analysis to score γ-H2AX induction, and its suitability to distinguish a radiosensitive (RS) cell line from a radioresistant (RR) one.

Materials and methods: γ-H2AX analyses were performed by semi-automated and automated microscopic methods and by flow cytometry before and after irradiation in two human lymphoblastoid cell lines and in lymphocytes from three healthy donors.

Results: Reproducible results were obtained by all the methodologies tested, although not all showed the same sensitivity. The RS cell line always showed higher foci counts and higher levels of immunofluorescence intensity after irradiation than the RR cell line.

Conclusions: Our results suggest that microscopic methodologies with z-stage capacity give the most accurate results after 1?Gy irradiation. However, for high doses of ionizing radiation, flow cytometry gives reliable results. Further studies will be necessary to determine the usefulness of γ-H2AX analysis to predict adverse side reactions in radiotherapy patients.     

甲氰咪胍对非酒精性脂肪性肝炎大鼠脂质过氧化作用的影响

目的：探讨甲氰咪胍对非酒精性脂肪性肝炎（NASH）大鼠脂质过氧化作用的影响。方法：雄性SD大鼠30只，随机分为3组，正常对照组、模型组、甲氰咪胍组，正常组普通饲料喂养，模型组喂高脂饮食，甲氰咪胍组在高脂饮食12w后给予甲氰咪胍灌胃治疗。16w末处死各组大鼠，测定血清转氨酶（ALT、AST）、血脂（TC、TG）、肝匀浆丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性，同时测定细胞色素P450（CYP450）和细胞色素2E1（CYP2E1）含量，光镜下观察肝组织学改变。结果：与正常组比较，模型组大鼠血清TG、TC水平，肝组织脂质过氧化产物MDA含量明显增高，而抗氧化物SOD活性明显降低，肝脏的脂肪变性程度和炎症活动度均显著增高，细胞色素P450和CYP2E1含量显著增高。甲氰咪胍组各项指标较模型组有明显改善（P〈0．05）。结论：甲氰咪胍通过抑制CYP2E1表达与其密切相关的脂质过氧化作用，改善NASH大鼠脂肪变性，减轻炎症反应。     

Histological and biochemical analysis of DNA damage after BNCT in rat model

To understand the mechanism of tumor cell death induced by boron neutron capture therapy (BNCT) and to optimize BNCT condition, we used rat tumor graft models and histological and biochemical analyses were carried out focusing on DNA damage response. Rat lymphosarcoma cells were grafted subcutaneously into male Wister rats. The rats with developed tumors were then treated with neutron beam irradiation 45 min after injection of 330 mg/kg bodyweight boronophenylalanine (¹⁰BPA) (+BPA) or saline control (–BPA). BNCT was carried out in the National Nuclear Center of the Republic of Kazakhstan (neutron flux: 1×10⁹ nvt/s, fluence: 6×10¹¹ nvt) with the presence of background γ-irradiation of 33 Gy. 6 and 20 h after BNCT treatment, tumors were resected, fixed and subjected to immunohistochemistry and biochemical analyses. Immunostaining of nuclei showed that double strand break (DSB) marker gamma H2AX staining was high in 20 h/+BPA sample but not in 20 h/–BPA samples. Poly(ADP-ribose), DSB and single strand break markers of DNA, also demonstrated this tendency. These two markers were observed at low levels in unirradiated tissues or 6 h after BNCT either under −BPA and +BPA conditions. HMGB1 level increased in 6 h/+BPA but not in 6 h/−BPA or 20 h/+BPA samples. The persistent staining of γH2AX and poly(ADP-ribose) in +BPA group suggests accumulated DSB damage after BNCT. The early HMGB1 upregulation and γH2AX and poly(ADP-ribose) observed later might be the markers for monitoring the DNA damage induced by BNCT.     

二咖啡酰奎宁酸抗肝纤维化及脂质过氧化作用的实验研究

目的 :观察二咖啡酰奎宁酸抗复合因素致大鼠肝纤维化及脂质过氧化作用。方法 :采用复合因素复制大鼠肝纤维化模型 ,以血清层连蛋白 (LN)、透明质酸 (HA)、丙二醛 (MDA)、一氧化氮 (NO)含量、肝组织光镜、电镜下形态学改变为观察内容 ,探讨二咖啡酰奎宁酸的治疗作用。结果 :二咖啡酰奎宁酸能够显著降低血清LN ,HA ,MDA ,NO含量 ,明显改善肝组织炎症及纤维化状态。结论 :二咖啡酰奎宁酸具有一定的抗肝纤维化及脂质过氧化作用     

gamma-H2AX foci formation in peripheral blood lymphocytes of tumor patients after local radiotherapy to different sites of the body: Dependence on the dose-distribution,irradiated site and time from start of treatment

Purpose: To evaluate the relationship between an estimated integral total body radiation dose delivered and phosphorylated histone H2AX protein (γ-H2AX) foci formation in peripheral blood lymphocytes of cancer patients.

Material and methods: γ-H2AX formation was quantified as the mean number of foci per lymphocyte (N_meanH2AX) and the percentage of lymphocytes with ≥n foci. The integrated total body radiation dose was estimated from the dose volume histogram of patient's body corrected for the proportion of the body scanned by computed tomography for 3D treatment planning.

Results: There was a strong linear correlation between the mean number of γ-H2AX foci per lymphocyte in the peripheral blood sample and integrated total body radiation dose (r = 0.83, p < 0.0001). The slope of the relationship was dependent on the site of body irradiated. In comparison to chest irradiation with a slope of 8.7 ± 0.8 foci Gy^?1, the slopes for brain, upper leg and pelvic sites were significantly shallower by ?4.7, ?4.3, and ?3.8 Gy^?1, respectively (p < 0.0001), while the slope for upper abdomen irradiation was significantly larger by 9.1 ± 2.6 Gy^?1 (p = 0.0007). There was a slight time effect since the start of radiotherapy on the slopes of the in vivo dose responses leading to shallower slopes (?1.5 ± 0.7 Gy^?1, p = 0.03) later (≥10 day) during radiotherapy. After in vitro irradiation, lymphocytes showed 10.41 ± 0.12 foci per Gy with no evidence of inter-individual heterogeneity.

Conclusions: γ-H2AX measurements in peripheral lymphocytes after local radiotherapy allow the estimation of the applied integral body dose. The site and time dependence have to be considered.     

Arginine glutamate improves healing of radiation-induced skin ulcers in guinea pigs

Abstract

Purpose: The increase in the incidence of the radiation-induced skin injury cases and the absence of standard treatments escalate the interest in finding new and effective drugs for these lesions. We studied the effect of a 40% solution of arginine glutamate on the healing of radiation-induced skin ulcers in guinea pigs.

Materials and methods: Radiation skin injury was produced on the thigh of guinea pigs by 60 Gy local X-ray irradiation. Treatment was started 6 weeks after the irradiation when ulcers had been formed. Arginine glutamate was administered by subcutaneous injections around the wound edge. Methyluracil was chosen as the comparison drug. The animals were sacrificed on day 21 after the start of treatment and the irradiated skin tissues were subjected to histological evaluation, cytokines analysis, lipid peroxidation and antioxidant enzymes analysis.

Results: We have shown that arginine glutamate significantly (p < 0.05) decreased levels of pro-inflammatory cytokines in the wound, restored the balance between lipid peroxidation formation and antioxidant enzymes activity and promoted cell proliferation as well as collagen synthesis.

Conclusions: These results demonstrate that arginine glutamate successfully improves the healing of radiation-induced skin ulcers. In all probability, the curative effect is associated with the interaction of arginine with nitric oxide synthase II and arginase I, but further investigations are needed to validate this.     

几丁糖在兔角膜碱烧伤中的抗氧化损伤作用

目的 探讨20g/L医用几丁糖在角膜碱烧伤中对脂质过氧化损伤、炎症反应的影响。方法 以15只新西兰大白兔制作兔角膜碱烧伤模型，设立几丁糖治疗组（右眼）和对照组（左眼）。于烧伤后第7，14，28天各取两组5只兔的角膜和房水检测超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量，并观察角膜组织病理学变化。结果 医用几丁糖治疗组较对照组同期SOD活性高，MDA生成量少，角膜炎症反应轻。结论 几丁糖在角膜碱烧伤急性期治疗中具有较强的抗脂质过氧化损伤、抑制炎症及促进愈合的作用。     

snoRNA在电离辐射诱导的DNA损伤中的作用及研究进展

核仁小RNA (small nucleolus RNA,snoRNA)是一类在真核生物中普遍存在的非编码RNA,根据其结构特征,可分为两大类:box C/D snoRNA和box H/ACA snoRNA,分别介导RNA 2''-O-甲基化和假尿嘧啶化修饰。近期研究发现,snoRNA还影响mRNA前体(pre-mRNA)的可变剪接,能够通过生成miRNA来介导基因沉默,并通过与蛋白质相互作用调节其功能活性。在电离辐射的生物效应中,DNA损伤与修复过程是至关重要的生物学基础。然而,目前关于snoRNA在电离辐射引起的DNA损伤中的作用的研究还相当有限。本综述旨在概述snoRNA的生物学功能及其在电离辐射诱导的DNA损伤修复中可能的调控角色,以期为探究snoRNA的功能及机制提供新思路。     

Development of a method for assessing non-targeted radiation damage in an artificial 3D human skin model

Purpose : Radon risks derive from exposure of bronchio-epithelial cells to high-linear energy transfer (LET) α -particles. α -particle exposure can result in bystander effects, where irradiated cells emit signals resulting in damage to nearby unirradiated bystander cells. This can result in non-linear dose-response relations, and inverse dose-rate effects. Domestic radon risk estimates are currently extrapolated from miner data, which are at both higher doses and higher dose-rates, so bystander effects on unhit cells could play a large role in the extrapolation of risks from mines to homes. Therefore, we extend an earlier quantitative mechanistic model of bystander effects to include protracted exposure, with the aim of quantifying the significance of the bystander effect for very prolonged exposures. Materials and methods : A model of high-LET bystander effects, originally developed to analyse oncogenic transformation in vitro, is extended to low dose-rates. The model considers radiation response as a superposition of bystander and linear direct e It attributes bystander effects to a small subpopulation of hypersensitive cells, with the bystander contribution dominating the direct contribution at very low acute doses but saturating as the dose increases. Inverse dose-rate effects are attributed to the replenishment of the hypersensitive subpopulation during prolonged irradiation. Results : The model was fitted to dose- and dose-rate-dependent radon-exposed miner data, suggesting that one directly hit target bronchio-epithelial cell can send bystander signals to about 50 neighbouring target cells. The model suggests that a naïve linear extrapolation of radon miner data to low doses, without accounting for dose-rate, would result in an underestimation of domestic radon risks by about a factor of 4, a value comparable with the empirical estimate applied in the recent BEIR-VI report on radon risk estimation. Conclusions : Bystander effects represent a plausible quantitative and mechanistic explanation of inverse dose-rate effects by high-LET radiation, resulting in non-linear dose-response relations and a complex interplay between the effects of dose and exposure time. The model presented provides a potential mechanistic underpinning for the empirical exposure-time correction factors applied in the recent BEIR-VI for domestic radon risk estimation.     

Application of postmortem lipid peroxidation in heart tissue to the diagnosis of myocardial damage

The response by the myocardial tissue to injury may be manifested as macroscopical or microscopical lesions or, following an ischemia-reperfusion process, by the formation of reactive oxidant substances (ROS) which, in turn, may increase lipid peroxidation levels in myocardial tissue. The aim of this study was to determine whether the levels of lipid peroxidation in myocardial tissue from subjects who had died from different causes are related to cardiac lesions of ischemic or traumatic aetiology. We studied 825 hypostasis-free samples taken from the myocardial tissue of 75 cadavers. In all cases, a survival period was known to have existed. Lipid peroxidation in myocardial tissue was estimated by the colorimetric determination of malondialdehyde. The highest levels of tissue peroxidation were observed in the group of subjects who died from myocardial infarction. We observed a statistically significant correlation between the extent of peroxidation and the presence of myocardial damage of ischemic or traumatic aetiology. In our study, lipid peroxidation was associated with myocardial injury. The data suggest that the extent of lipid peroxidation in myocardial tissue may be used as a reliable indicator of myocardial damage in support of data obtained by conventional microscopy.     

P53，Bax,Bcl—2蛋白表达及细胞凋亡在急性放射性皮肤溃疡发生发展过程中的作用探讨

目的：研究细胞凋及一些凋亡相关基因（p53,bcl-2,bax)的表达在急性放射性皮肤贵疡发生发展过程中的作用,方法：采用Wistar大鼠以^60Cor射线进行局部照射,建立急性放射性皮肤溃疡动物模型,观察病变40d,然后采用免疫组化方法检测皮肤溃疡组织中P53,Bcl-2,Bax蛋白表达,并采用原位末端标记法（TUNEL）检测细胞凋亡,结果,照后14d照射野内开始出现皮肤细胞和小血管平滑肌中,照后14－21d为Bax蛋白表达高峰,之后逐渐减弱,主要定位于血管内皮细胞,部分成纤维细胞及新生表皮细胞中,Bcl-2则在照后1－11d呈弱或中度阳性,定位于表皮,毛囊上皮及血管内皮口,之后为阴性或可疑阳性,照后11－35d,上述细胞特别是血管内皮细胞凋亡率较正常伤口愈合早期增高,结论：辐射诱导的P53,Bax-Bcl-2表达的变化及细胞亡率特别是血管内皮细胞凋亡率的增主屿放射性皮肤溃疡发生,发展及难愈合（不能形成有效肉芽组织）的分子机制相关。     

Clemens von Sonntag and the early history of radiation-induced sugar damage in DNA

Abstract

Purpose: This article reviews the early history of ionizing radiation-induced sugar damage in DNA in dedication to Prof. Clemens von Sonntag, who recently passed away. It covers the time between 1968 and 1978, during which most of the work on the ionizing radiation-induced damage to polyalcohols, carbohydrates and the 2?-deoxyribose moiety in DNA was performed. Methodologies using gas chromatography-mass spectrometry (GC-MS) were developed to identify and quantify the radiation-induced products that had previously remained elusive. Products were identified by GC-MS either directly or after reduction of samples with NaBH₄ or NaBD₄. Incorporation of deuterium atoms by NaBD₄-reduction facilitated the identification of aldehyde, keto, carboxyl and deoxy groups in the molecules. Numerous products of a polyalcohol and carbohydrates were identified and quantified. Mechanisms of product formation were proposed. Several products of the 2?-deoxyribose moiety in DNA were identified, indicating that they were released from DNA strand, not bound to it. Alkali labile sites and products still remaining within DNA or bound to DNA as end groups were also elucidated by first reducing irradiated samples with NaBD₄ followed by alkali treatment and GC-MS analysis.

Conclusion: The knowledge of the products of the 2?-deoxyribose moiety in DNA led to the first mechanistic understanding of various pathways of hydroxyl radical-induced DNA strand breakage. To this date, some of these mechanisms still remain the most-widely studied mechanisms of DNA damage. Prof. von Sonntag's contributions to the understanding of the radiation chemistry of carbohydrates and DNA helped shape this field of science for years to come.     

Induction of radical scavenging ability and suppression of lipid peroxidation in rat liver microsomes following whole-body,low-dose X-irradiation

Purpose: To investigate changes in radical scavenging ability and lipid peroxidation in liver microsomal membranes and cooperative suppression of lipid peroxidation by microsomal and cytosolic radical scavengers, 24 h after whole-body, low-dose X-irradiation of rats.

Materials and methods: Male Wistar rats were irradiated with 1 – 50 cGy of X-rays. Liver microsomal radical scavenging ability was determined using the trapping ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH), a stable free radical. Microsomal α-tocopherol (Vit.E) content was determined using an electrochemical detector. Microsomal glutathione peroxidase (GPx) activity was determined as the consuming rate of NADPH. Microsomal lipid peroxidation was determined by the thiobarbituric acid method.

Results: Low molecular weight radical scavenging ability of rat liver microsomes increased 24 h after whole-body, low-dose X-irradiation when α-tocopherol was included, showing a maximum level at 5 – 10 cGy. Microsomal GPx activity also increased 24 h after 5 cGy irradiation. The lipid peroxidation level in microsomes decreased, showing a maximal suppression at 5 cGy. High-dose irradiation-induced microsomal lipid peroxidation was strongly suppressed cooperatively by microsomal and cytosolic antioxidants induced by low-dose irradiation.

Conclusion: Low doses of radiation induce increases in liver microsomal antioxidants, which in turn result in enhanced suppression of microsomal lipid peroxidation cooperatively with cytosolic antioxidants induced by low-dose irradiation.     

Assessment of squalene eligibility in bettering some maternal and fetal disorders instigated by gamma irradiation of rats at mid gestation

Purpose: Squalene is an eminent vital part of the synthesis of steroid hormones in the body as well as the first specific intermediate in cholesterol biosynthesis that plays an essential role in normal embryogenesis. The present work was designed to test the maternal and embryonic response to the modulating capacity of squalene (0.4?ml/kg/d), when supplemented to rats from days 1 to 18 of pregnancy, against the damaging consequences induced by maternal subjection to 3 Gy gamma irradiation on day 10 post-conception.

Materials and methods: The experimental protocol comprised of four different pregnant groups, namely: (1) control, (2) squalene supplemented, (3) irradiated and (4) squalene supplemented?+ irradiated.

Results: It has been detected that radiation has increased the maternal blood lactate dehydrogenase (as a marker of tissue injury), cholesterol, triglycerides, estradiol and progesterone and has also provoked the oxidative stress that has been demonstrated by the increased malondialdehyde (MDA) and the decreased glutathione (GSH) and superoxide dismutase (SOD). These maternal changes were associated with high embryonic lethality, growth retardation, severe developmental abnormalities and defective neural tube closure expressed by exencephaly. However, squalene treatment has significantly improved the radiation imposed maternal variations and reduced the embryonic mortality, although it has not been able to attenuate the embryonic neural tube defects.

Conclusions: It has been presumed that the maternal mid-gestational irradiation (day 10) has affected the fetal nervous system development with concomitant maternal oxidative stress, hyperlipidemia, and increased progesterone and estradiol levels. Squalene uptake has improved the maternal variations and reduced the embryonic mortality while could not stop or improve the embryonic neural tube defects imposed by radiation at this exact radiation timing.