用脉冲电场凝胶电泳检测电离辐射所致哺乳动物细胞DNA双链断裂

 本文将反向交变电场和六角形电极电场这两种脉冲电场凝胶电泳技术应用于X线照射小鼠乳癌细胞SR-1所致DNA双链断裂的检测,在本实验条件下,用这种电泳都能检测到低至1.5Gy照射所产生的DNA双链断裂,并且用六角形电极电场电泳获得了DNA双链断裂程度与照射剂量之间的良好线性关系,此外,还用此方法观察了不同浓度自由基清除剂DMSO对X线照射SR-1细胞所致DNA双链断裂的保护作用,结果进一步证实本方法的可靠性。     

衰老过程中大白鼠脾细胞的DNA单链断裂与重接能力的变化

 DNA被紫外线损伤后,由DNA切除修复酶切除嘧啶二聚体,随之以另一条正常的DNA链为模板修复合成DNA片段,最后由DNA连接酶将新合成的DNA片与原有的DNA链连接。本文用荧光法测定DNA修复过程中DNA单链的断裂及重接能力与衰老的关系。结果表明,不同年龄大鼠脾细胞均具有修复DNA单链断裂的能力,DNA单链断裂重接的能力与年龄有相关性,断乳鼠及青年鼠的脾细胞当保温至30min时,即开始了DNA链的重接,保温90min后则恢复到原有水平;而老年鼠脾细胞保温至90min时才开始DNA链的重接,保温150min,尚未恢复到原有水平。还发现,断乳鼠及老年鼠脾细胞的单链DNA含量高于青年鼠。     

Histochemical Demonstration of DNA Double Strand Breaks by in Situ 3'-tailing Reaction in Apoptotic Endometrium

A new histochemical technique, called in situ 3'-tailing reaction (ISTR), to detect DNA double strand breaks (DSB) was developed and applied to tissue sections of apoptotic endometrium. To demonstrate DSB, biotin-labeled and unlabeled dATPs with terminal deoxynucleotidyl transferase (TdT) were added to the many 3-hydroxyl termini of DNA fragments generated in the apoptotic cells. For an efficient 3'-end labeling, it was necessary to treat the sections with λ-exonuclease (λEx) prior to the TdT reaction to generate 3'-protruding ends. The λEx-TdT reaction specifically labeled nuclear fragments in the apoptotic cells in paraformaldehyde fixed frozen sections. In paraffin sections, pretreatment with proteinase K was effective for 3'-tailing reaction. ISTR should be a useful tool for detecting dying cells in both physiological and pathological states.     

三硝基甲苯致晶状体上皮细胞DNA损伤作用的研究

本文通过出生后７天和２４个月两组大鼠晶状体与２０％三硝基甲苯（ＴＮＴ）甘油：水（８：２）混悬液体外培养,培养箱通５％ＣＯ２,恒温３７℃,观察ＴＮＴ对晶状体上皮细胞ＤＮＡ的损伤程度,结果以反映单链ＤＮＡ断裂强度的Ｆ６３０／Ｆ５３０比值表示。ＨＰＬＣ分析发现经体外ＴＮＴ孵育的晶状体内含有ＴＮＴ及其代谢产物;当共同培养９６ｈ后,Ｆ６４０／Ｆ５３０比值随着ＴＮＴ剂量增加而增大,达到４０μＬ（１１．７４μｍｏｌ／Ｌ）时趋向平稳;当用ＴＮＴ浓度为４．４０μｍｏｌ／Ｌ的相同剂量培养时,４８小时后单链ＤＮＡ断裂最为严重Ｆ６３０／Ｆ５３０比值为１．５０,明显高子正常对照组（Ｐ＜０．０１）;幼鼠单链ＤＮＡ断裂程度显著高于老龄鼠（Ｐ＜０．０１）。     

DNA Single Strand Breaks by Aromatic Nitroso Compounds in the Presence of Thiols

Aromatic nitroso compounds, nitrosobenzene (NB), N, N-dimethyl-4-nitrosoaniline (DMNA) and 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), caused DNA single strand breaks in the presence of thiol compounds. The strand breaking was inhibited completely by free radical scavenger ethanol. Electron spin resonance (ESR) studies showed that hydronitroxyl (or sulfur-substituted nitroxyl) radicals were generated in the early stage of the interactions. Formation of these radicals was not inhibited by ethanol, indicating that these radicals did not directly contribute to the strand breaking. The DNA strand breaking was inhibited partially by superoxide dismutase and catalase under the limited conditions, but not by removal of oxygen from or addition of metal chelators to the reaction mixture. By ESR-spin trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the DMPO-OH spin adduct was detected. Formation of the spin adduct was inhibited by superoxide dismutase and catalase. The hydronitroxyl (or the sulfur-substituted nitroxyl) radicals may reduce oxygen into active oxygen species and also transformed by themselves into other unidentified free radical species to cause the DNA strand breaks.     

3-氨基苯酰胺对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老和凋亡有着密切的关系.在各种DNA损伤中,单链断裂(single-strand breaks, SSBs)是最常见的类型之一,既可直接通过内源活性氧或离子化辐射产生,也可间接地在DNA代谢或碱基切除修复期间产生.已知多聚(ADP-核糖)聚合酶［poly(ADPribose) polymerase, PARP］在SSBs修复中起着极为重要的作用.本实验观察了PARP抑制剂3-氨基苯酰胺(3-aminobenzamide, 3-AB)对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接的效应以及对过氧化氢(H2O2)抑制HeLa细胞增殖的影响.结果表明3-AB能够显著地抑制氧化应激诱导的HeLa细胞端粒DNA链断裂后的重连接作用,并能增强H2O2对HeLa细胞增殖的抑制作用,提示PARP参与了端粒DNA链断裂损伤的修复过程.     

Contrasting effects of Krüppel‐like factor 4 on X‐ray‐induced double‐strand and single‐strand DNA breaks in mouse astrocytes

Astrocytes, the most common cell type in the brain, play a principal role in the repair of damaged brain tissues during external radiotherapy of brain tumours. As a downstream gene of p53, the effects of Krüppel‐like factor 4 (KLF4) in response to X‐ray‐induced DNA damage in astrocytes are unclear. In the present study, KLF4 expression was upregulated after the exposure of astrocytes isolated from the murine brain. Inhibition of KLF4 expression using lentiviral transduction produced less double‐strand DNA breaks (DSB) determined by a neutral comet assay and flow cytometric analysis of phosphorylated histone family 2A variant and more single‐strand DNA breaks (SSB) determined by a basic comet assay when the astrocytes were exposed to 4 Gy of X‐ray radiation. These data suggest that radiation exposure of the tissues around brain tumour during radiation therapy causes KLF4 overexpression in astrocytes, which induces more DSB and reduces SSB. This causes the adverse effects of radiation therapy in the treatment of brain tumours. Copyright © 2013 John Wiley & Sons, Ltd.     

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.     

Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells

Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects of ¹³⁷Cs γ-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation, and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired 24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.     

激光辐照对离体质粒DNA的断裂效应

利用低功率的Ｈｅ－ Ｎｅ 激光辐照离体质粒ＤＮＡ,并用离子束和紫外线为对照,分析了质粒ＤＮＡ的单双链断裂效应。结果表明：Ｈｅ－ Ｎｅ 激光辐照可诱发质粒ＤＮＡ 断裂,且断裂频率随辐照剂量增大而提高;Ｈｅ－ Ｎｅ 激光辐照后的质粒超螺旋双链ＤＮＡ存活率剂量曲线不同于紫外线和氮离子束。Ｈｅ－ Ｎｅ 激光诱发双链断裂的频率低于氮离子束和紫外线。     

TNT体外大鼠白内障模型中的DNA单链断裂及其重接修复的研究

在大鼠晶状体器官培养的条件下,运用单细胞电泳法（ＳＣＧ）,对远在晶状体混浊之前的晶状体上皮细胞进行了有关ＴＮＴ致其ＤＮＡ损伤（ＳＳＢ）与修复的初步观察,提示ＤＮＡ损伤也是体外ＴＮＴ性白内障中的早期变化．     

Early Detection of DNA Strand Breaks in the Brain After Transient Focal Ischemia: Implications for the Role of DNA Damage in Apoptosis and Neuronal Cell Death

Abstract: Using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single-strand breaks (SSBs) detected by PANT were present in neurons after as little as 1 min of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double-strand breaks (DSBs) detected by TUNEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB-containing cells was greater than that of DSB-containing cells at all time points tested. SSB-containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16–72 h of reperfusion, many SSB- and DSB-containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis.     

A reporter mouse for in vivo detection of DNA damage in embryonic germ cells

Maintaining genome integrity in the germline is essential for survival and propagation of a species. In both mouse and human, germ cells originate during fetal development and are hypersensitive to both endogenous and exogenous DNA damaging agents. Currently, mechanistic understanding of how primordial germ cells respond to DNA damage is limited in part by the tools available to study these cells. We developed a mouse transgenic reporter strain expressing a 53BP1‐mCherry fusion protein under the control of the Oct4ΔPE embryonic germ cell‐specific promoter. This reporter binds sites of DNA double strand breaks (DSBs) on chromatin, forming foci. Using ionizing radiation as a DNA DSB‐inducing agent, we show that the transgenic reporter expresses specifically in the embryonic germ cells of both sexes and forms DNA damage induced foci in both a dose‐ and time‐dependent manner. The dynamic time‐sensitive and dose‐sensitive DNA damage detection ability of this transgenic reporter, in combination with its specific expression in embryonic germ cells, makes it a versatile and valuable tool for increasing our understanding of DNA damage responses in these unique cells.     

Microwaves from UMTS/GSM mobile phones induce long-lasting inhibition of 53BP1/gamma-H2AX DNA repair foci in human lymphocytes

We have recently described frequency-dependent effects of mobile phone microwaves (MWs) of global system for mobile communication (GSM) on human lymphocytes from persons reporting hypersensitivity to electromagnetic fields and healthy persons. Contrary to GSM, universal global telecommunications system (UMTS) mobile phones emit wide-band MW signals. Hypothetically, UMTS MWs may result in higher biological effects compared to GSM signal because of eventual "effective" frequencies within the wideband. Here, we report for the first time that UMTS MWs affect chromatin and inhibit formation of DNA double-strand breaks co-localizing 53BP1/gamma-H2AX DNA repair foci in human lymphocytes from hypersensitive and healthy persons and confirm that effects of GSM MWs depend on carrier frequency. Remarkably, the effects of MWs on 53BP1/gamma-H2AX foci persisted up to 72 h following exposure of cells, even longer than the stress response following heat shock. The data are in line with the hypothesis that the type of signal, UMTS MWs, may have higher biological efficiency and possibly larger health risk effects compared to GSM radiation emissions. No significant differences in effects between groups of healthy and hypersensitive subjects were observed, except for the effects of UMTS MWs and GSM-915 MHz MWs on the formation of the DNA repair foci, which were different for hypersensitive (P < 0.02[53BP1]//0.01[gamma-H2AX]) but not for control subjects (P > 0.05). The non-parametric statistics used here did not indicate specificity of the differences revealed between the effects of GSM and UMTS MWs on cells from hypersensitive subjects and more data are needed to study the nature of these differences.     

Involvement of Hydroxyl Radical Formation and Dna Strand Breakage in the Cytotoxicity of Anthraquinone Antitumour Agents

Four 9,10-anthraquinones (AQ) mono- or bis-substituted with the -NH(CH₂)₂ NH(CH₂)₂OH group were studied. 1-AQ, 1,5-AQ and 1,8-AQ but not 1,4-AQ (100°M) generated pBR322 plasmid DNA single strand breaks in the presence of purified NADPH dependent cytochrome P450 reductase. 1-AQ, 1,5-AQ and 1,8-AQ (at 100 °M) stimulated hydroxyl radical formation in MCF-7 S9 cell fraction (as measured by dimethyl pyrolline N-oxide spin trapping) and MCF-7 DNA strand breaks as measured by alkaline filter elution. In contrast 1,4-AQ did not stimulate hydroxyl radical formation and produced considerably less strand breaks in MCF-7 cells compared to the other AQ's. It would appear that the position of the -NH(CH₂)₂ NH(CH₂)₂OH groups on the chromophore is an important determinant in the metabolic activation of cytotoxic anthraquinones. This may contribute to the cytotoxicity (ID₅₀ values) of 1-AQ (0.06 °M), 1-8-AQ (0.5 °M) and 1,5-AQ (12.3 °M) but not the 1,4-AQ (1.2 °M).     

Involvement of Hydroxyl Radical Formation and Dna Strand Breakage in the Cytotoxicity of Anthraquinone Antitumour Agents

Four 9,10-anthraquinones (AQ) mono- or bis-substituted with the -NH(CH₂)₂ NH(CH₂)₂OH group were studied. 1-AQ, 1,5-AQ and 1,8-AQ but not 1,4-AQ (100°M) generated pBR322 plasmid DNA single strand breaks in the presence of purified NADPH dependent cytochrome P450 reductase. 1-AQ, 1,5-AQ and 1,8-AQ (at 100 °M) stimulated hydroxyl radical formation in MCF-7 S9 cell fraction (as measured by dimethyl pyrolline N-oxide spin trapping) and MCF-7 DNA strand breaks as measured by alkaline filter elution. In contrast 1,4-AQ did not stimulate hydroxyl radical formation and produced considerably less strand breaks in MCF-7 cells compared to the other AQ's. It would appear that the position of the -NH(CH₂)₂ NH(CH₂)₂OH groups on the chromophore is an important determinant in the metabolic activation of cytotoxic anthraquinones. This may contribute to the cytotoxicity (ID₅₀ values) of 1-AQ (0.06 °M), 1-8-AQ (0.5 °M) and 1,5-AQ (12.3 °M) but not the 1,4-AQ (1.2 °M).     

Time-varying magnetic fields of 60 Hz at 7 mT induce DNA double-strand breaks and activate DNA damage checkpoints without apoptosis

The potential genotoxic effect of a time-varying magnetic field (MF) on human cells was investigated. Upon continuous exposure of human primary fibroblast and cervical cancer cells to a 60 Hz MF at 7 mT for 10-60 min, no significant change in cell viability was observed. However, deoxyribonucleic acid (DNA) double-strand breaks (DSBs) were detected, and the DNA damage checkpoint pathway was activated in these cells without programmed cell death (called apoptosis). The exposure of human cells to a 60 Hz MF did not induce intracellular reactive oxygen species (ROS) production, suggesting that the observed DNA DSBs are not directly caused by ROS. We also compared the position and time dependency of DNA DSBs with numerical simulation of MFs. The Lorentz force and eddy currents in these experiments were numerically calculated to investigate the influence of each factor on DNA DSBs. The DNA DSBs mainly occurred at the central region, where the MF was strongest, after a 30-min exposure. After 90 min, however, the amount of DNA DSBs increased rapidly in the outer regions, where the eddy current and Lorentz force were strong.     

Inhibition of polarity-regulating kinase PAR1b contributes to Helicobacter pylori inflicted DNA Double Strand Breaks in gastric cells

The serine/threonine kinase Par1 is a core component of the machinery that sets up polarity in the embryo and regulates cell fate decisions but its role in the homeostasis of adult tissues is poorly understood. Inhibition of Par1 by the bacterium Helicobacter pylori (H. pylori) represents the only established pathology that affects Par1 function in an adult epithelium. Thus, during chronic H. pylori infection of the gastric mucosa Par1 is one of the targets of the non-obligate H.pylori cytotoxic protein and oncogene CagA, which stimulates inflammation and triggers morphological changes, both believed to contribute to the gastric cancer risk imposed by H. pylori infection. Based on Par1’s role in cell polarity, it has been speculated that Par1 inhibition affects epithelial polarity. Here we report the unexpected finding that CagA-mediated Par1-inhibition promotes the generation of DNA Double Strand Breaks in primary gastric epithelial cells, which likely contributes to the reported accumulation of mutations in chronically infected mucosal cells.

Abbreviations: AGS: human gastric adenocarcinoma cell line; CM: CagA Multimerization (and Par1 binding) domain; H. pylori: Helicobacter pylori; DSB: Double Strand Break; HGECs: human (primary) gastric epithelial cells; IB: immunoblot; IF: immunofluorescence; MOI: Multiplicity of Infection; ROS: reactive oxygen species; Par1: Partitioning Defective 1 kinase; WT: wild type     

An Extremely Low Frequency Magnetic Field and Global Cerebral Ischemia Affect Pituitary ACTH and TSH Cells in Gerbils

The neuroendocrine system can be modulated by a magnetic field and cerebral ischemia as external and internal stressors, respectively. This study deals with the separate or combined effects of an extremely low frequency (ELF) magnetic field (50 Hz, average magnetic field of 0.5 mT) for 7 days and global cerebral ischemia for 10 min on the morpho-functional features of pituitary adrenocorticotrophic (ACTH) and thyrotrophic (TSH) cells in 3-month-old gerbils. To determine the immediate and delayed effects of the applied stressors, measurements were made on the 7th and 14th days after the onset of the experiment. The ELF magnetic field and 10-min global cerebral ischemia, separately and particularly in combination, decreased (P < 0.05) the volume density of ACTH cells, while only in combination were intracellular ACTH content and plasma ACTH concentration increased (P < 0.05) on day 7. The ELF magnetic field elevated serum TSH concentration on day 7 and intracellular TSHβ content on day 14 (P < 0.05). Also, 10-min global cerebral ischemia alone increased serum TSH concentration (P < 0.05), while in combination with the ELF magnetic field it elevated (P < 0.05) intracellular TSHβ content on day 14. In conclusion, an ELF magnetic field and/or 10-min global cerebral ischemia can induce immediate and delayed stimulation of ACTH and TSH synthesis and secretion. Bioelectromagnetics. 2020;41:91–103. © 2019 Bioelectromagnetics Society.