The comet assay for DNA damage and repair

The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.     

An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels

A procedure for quick and simple elution of DNA from agarose gels is presented. After electrophoresis, bands of interest are cut out of the gel and the slices are equilibrated in a neutral salt buffer. The slices are then frozen and centrifuged through a filtration assembly whereby the DNA-containing buffer is squeezed out. The method is simple, quick, and suitable for the safe handling of small amounts of DNA (less than 1 microgram). The isolated DNA is susceptible to any enzymatic reaction and also to chemical sequencing. The method is most useful for rapid preparation of specifically end-labeled DNA fragments (e.g., for sequencing), but may also be utilized for any other preparative applications.     

Antioxidative effects of fluvastatin and its metabolites against oxidative DNA damage in mammalian cultured cells

We investigated the effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on reactive oxygen species (ROS) and on oxidative DNA damage in vitro, as well as the effects of the main fluvastatin metabolites (M2, M3, and M4) and other inhibitors of the same enzyme, pravastatin and simvastatin. The hydroxyl radical and the superoxide anion scavenging activities of fluvastatin and its metabolites were evaluated using an electron spin resonance spectrometer. Fluvastatin and its metabolites showed superoxide anion scavenging activity in the hypoxanthine-xanthine oxidase system and a strong scavenging effect on the hydroxyl radical produced from Fenton's reaction. Protective effects of fluvastatin on ROS-induced DNA damage of CHL/IU cells were assessed using the single-cell gel electrophoresis assay. CHL/IU cells were exposed to either hydrogen peroxide or t-butylhydroperoxide. Fluvastatin and its metabolites showed protective effects on DNA damage as potent as the reference antioxidants, ascorbic acid, trolox, and probucol, though pravastatin and simvastatin did not exert clear protective effects. These observations suggest that fluvastatin and its metabolites may have radical scavenging activity and the potential to protect cells against oxidative DNA damage. Furthermore, ROS are thought to play a major role in the etiology of a wide variety of diseases such as cellular aging, inflammation, diabetes, and cancer development, so fluvastatin might reduce these risks.     

The effects of solar ultraviolet-B radiation on the growth and yield of barley are accompanied by increased DNA damage and antioxidant responses

There is limited information on the impacts of present-day solar ultraviolet-B radiation (UV-B) on biomass and grain yield of field crops and on the mechanisms that confer tolerance to UV-B radiation under field conditions. We investigated the effects of solar UV-B on aspects of the biochemistry, growth and yield of barley crops using replicated field plots and two barley strains, a catalase (CAT)-deficient mutant (RPr 79/4) and its wild-type mother line (Maris Mink). Solar UV-B reduced biomass accumulation and grain yield in both strains. The effects on crop biomass accumulation tended to be more severe in RPr 79/4 (≈ 32% reduction) than in the mother line (≈ 20% reduction). Solar UV-B caused measurable DNA damage in leaf tissue, in spite of inducing a significant increase in UV-absorbing sunscreens in the two lines. Maris Mink responded to solar UV-B with increased CAT and ascorbate peroxidase (APx) activity. No effects of UV-B on total superoxide dismutase (SOD) activity were detected. Compared with the wild type, RPr 79/4 had lower CAT activity, as expected, but higher APx activity. Neither of these activities increased in response to UV-B in RPr 79/4. These results suggest that growth inhibition by solar UV-B involves DNA damage and oxidative stress, and that constitutive and UV-B-induced antioxidant capacity may play an important role in UV-B tolerance.     

Isolation of plant DNA: A fast,inexpensive, and reliable method

We describe here a simple method to isolate DNA of high molecular weight from a wide variety of plant materials, such as trees, herbaceous plants, cell suspension cultures, calli, seeds, dried embryos, ferns and lichens. The crucial step of the extraction is the use of an acidic extraction medium. When necessary, the sample was separated on a fast RPC-5 column providing us with highly purified DNA suitable not only for restriction endonuclease analyses but also for PCR experiments, RLFP analyses, or detection of adducts.     

Effects of heavy metals Cd2+, Pb2+ and Zn2+ on DNA damage of loach Misgurnus anguillicaudatus

The effects of heavy metals Cd2+, Pb2+ and Zn2+ at 0.05, 0.5 and 5.0 mg/L level and their interactions at 0.5 mg/L level on DNA damage in hepatopancreas of loach Misgurnus anguillicaudatus for 1-35 days exposure were examined by single cell gel electrophoresis (SCGE). For each test group, 20 loaches with similar body size (5.17-7.99g; 11.79-13.21 cm) were selected and kept in aquaria with dechlori-nated water at (22±1)℃ and fed a commercial diet every 48 h. According to the percentage of damaged DNA with tail and its TL/D (tail length to diameter of nucleus) value, the relationship between DNA damage degree and heavy metal dose and exposure time was determined. Results showed that the percentage of damaged DNA and the TL/D value were increased with the prolonged exposure time. The highest percentage (84.85%) of damaged DNA was shown in 5.0 mg/L Zn2+ group after 28 days exposure and the biggest TL/D value (2.50) in all treated groups after 35 days exposure. During the first treated week, the damnification of DNA was mainly recognized as the first level, after that time, the third damaged level was mostly observed and the percentage of damaged DNA was beyond 80%. The joint toxic effects among Cd2+, Pb2+ or Zn2+ revealed much complexity, but it generally displayed that the presence of Cd2+ could enhance the genotoxicity of Pb2+ or Zn2+. In conclusion, the results suggested that there was a significant time-and dose-depended relationship between the heavy metal and DNA damage in hepatopancreas of loach, and SCGE could represent a useful means to evaluate the genotoxicity of environmental contamination on aquatic organisms.     

A simple method for isolation of high-molecular-weight DNA from Rhodotorula gracilis

Unsheared DNA has been isolated from Rhodotorula and Rhodosporidium yeasts using a cell-wall-digesting enzyme preparation from Paecilomyces lilacinus. Pulsed-field gel electrophoresis indicated that at least 11 chromosomes were present in Rhodot. gracilis ATCC 90950. The DNA was amenable to digestion with restriction enzymes.The authors are with the Department of Microbiology, Central Food Technological Research Institute, Mysore-570 013, India.     

Survivorship, development, and DNA damage in echinoderm embryos and larvae exposed to ultraviolet radiation (290-400 nm)

Laboratory experiments utilizing ecologically relevant irradiances of ultraviolet radiation (UVR) known to occur in shallow Gulf of Maine waters were conducted on the planktonic embryos and larvae of two common benthic echinoids; the green sea urchin Strongylocentrotus droebachiensis and the sand dollar Echinarachnius parma. Significant decreases in survivorship were observed in freshly fertilized embryos of both species with greater mortality in E. parma that was associated with the absence of UVR-absorbing compounds, the mycosporine-like amino acids. Experiments on blastula, gastrula, and prism larval stages of S. droebachiensis also showed significant decreases in survivorship, delays in development, and abnormal embryos and larvae associated with exposure to UVR. Additionally, all developmental stages of S. droebachiensis experimentally exposed to UVR resulted in significant increases in DNA damage, measured as cyclobutane pyrimidine dimer photoproducts. The observed delays in early cleavage and subsequent developmental stages for S. droebachiensis are correlated with DNA damage. It is postulated that cell cycle arrest at critical checkpoints after DNA damage, mediated by a suite of cell cycle genes, is a component of the observed UVR induced developmental delays.     

The effect of Helicobacter pylori infection on levels of DNA damage in gastric epithelial cells

Background. Helicobacter pylori infection leads to an increased risk of developing gastric cancer. The mechanism through which this occurs is not known. We aimed to determine the effect of H. pylori and gastritis on levels of DNA damage in gastric epithelial cells. Methods. Epithelial cells were isolated from antral biopsies from 111 patients. DNA damage was determined using single cell gel electrophoresis and the proportion of cells with damage calculated before and 6 weeks after eradication of H. pylori. Cell suspensions generated by sequential digestions of the same biopsies were assayed to determine the effect of cell position within the gastric pit on DNA damage. Results. DNA damage was significantly higher in normal gastric mucosa than in H. pylori gastritis [median (interquartile range) 65% (58.5–75.8), n = 18 and 21% (11.9–29.8), n = 65, respectively, p < .001]. Intermediate levels were found in reactive gastritis [55.5% (41.3–71.7), n = 13] and H. pylori negative chronic gastritis [50.5% (36.3–60.0), n = 15]. DNA damage rose 6 weeks after successful eradication of H. pylori[to 39.5% (26.3–51.0), p = .007] but was still lower than in normal mucosa. Chronic inflammation was the most important histological factor that determined DNA damage. DNA damage fell with increasing digestion times (r = –.92 and –.88 for normal mucosa and H. pylori gastritis, respectively). Conclusions. Lower levels of DNA damage in cells isolated from H. pylori infected gastric biopsies may be a reflection of increased cell turnover in H. pylori gastritis. The investigation of mature gastric epithelial cells for DNA damage is unlikely to elucidate the mechanisms underlying gastric carcinogenesis.     

An in vitro approach to study chromosomal DNA damage

In this study a simple electrophoresis approach has been proposed for assessing DNA damage per chromosome in vitro. Novel procedures of gel casting, sample loading, electrophoresis and quantification of damage have been suggested. Sets of Saccharomyces cerevisiae chromosomes subjected to DNA damage by Bleomycin, Co⁶⁰--radiation alone and in combination with Hoechst were studied in detail. Statistical analyses showed that damage induced by Bleomycin bore linear positive correlation with %GA (r=0.97) and %GT (r=0.61) contents of chromosomes. Samples pre-treated with Hoechst showed much less damage by Co⁶⁰--irradiation as compared to samples not treated with Hoechst but exposed to Co⁶⁰--irradiation. The `protective effect of Hoechst' bore linear positive correlation (r=0.8) with %TAT content of chromosomes.     

溴氰菊酯致大鼠DNA损伤及对肝功能的影响

目的：研究溴氰菊酯（DM）对大鼠外周血淋巴细胞DNA的损伤作用及对肝脏功能的影响。方法：32只雌性Wistar大鼠随机分成4组,染毒剂量分别为0,3．125,6．250,12．500mg／kg,连续灌胃染毒10天。DNA损伤采用单细胞凝胶电泳（彗星实验）进行评价,并测定丙氨酸氨基转移酶（ALT）以反映肝功能变化。结果：染毒组大鼠外周淋巴细胞的尾DNA％（Tail DNA％）、尾矩（Tail Moment）和Olive尾矩（Olive Tail Moment）均高于对照组（P〈0．05）,差别有统计学意义。各染毒组与对照组的肝功能差异均无统计学意义。结论：DM可导致外周血淋巴细胞DNA损伤。     

A Method for Isolation of Chloroplast DNA and Mitochondrial DNA from Sunflower

We present a method for isolation of chloroplast and mitochondrial DNA from sunflower seedlings. The protocol includes: organelle isolation, deoxyribonuclease treatment, lysis, deproteinisation and a final DNA purification with sodium dodecyl sulphate and potassium acetate. The organelle DNA yield is 5–10 micrograms per gram of tissue and the DNA is fully restrictable. The technique is inexpensive and appropriate for the isolation of multiple samples of organelle DNA from a small amount of tissue.     

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25±8.45 μm; non-smokers, 21.6±2.06 μm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5±20.34 μm; non-smokers, 79.2±11.59 μm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13±10.73 μm; non-smokers, (27.2±4.13 μm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.     

Radioadaptive response: Efficient repair of radiation-induced DNA damage in adapted cells

To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37°C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage.     

DNA断裂检测方法──单细胞凝胶电泳法

单细胞凝胶电泳(single cell gel electrophoresis assay,SCGE)也叫彗星试验(comet assay),是一种快速、敏感、简便、廉价的检测单个哺乳动物细胞DNA断裂的技术,目前已用于检测氧化、紫外线和电离辐射引起的损伤,以及三氯乙烷、丙烯酰胺等化学物及老化、吸烟所致损害的研究.文章介绍SCGE的发展、检测分析方法、原理及其在DNA损伤与修复、生物监测、遗传毒理研究、肿瘤治疗方案优化和疗效研究方面的应用前景．     

Identification of small nuclear DNA molecules of the cuban sugarcane cultivar Ja60-5 via pulsed field gel electrophoresis

We present evidence of the existence of at least 7 small nuclear DNA molecules in the Ja60-5 sugarcane cultivar genome. These small molecules were separated by means of transversal alternating field electrophoresis (TAFE); their sizes ranged from about 230–3000 kbp, and they hybridized with telomeric, subcentromeric, and various nuclear DNA probes. Here, we also report the use and the modifications of DNA from microorganisms, to immobilize nuclear DNA from sugarcane suitable for analysis via pulsed field gel electrophoresis. The protocol is simple, inexpensive, and easy to perform.     

Preparation of megabase-sized tomato DNA and separation of large restriction fragments by field inversion gel electrophoresis (FIGE)

The Schwartz and Cantor technique for releasing and fractionating megabase-sized DNA from agarose-embedded cells is beginning to bridge the gap in resoluation between classical genetics and current molecular DNA techniques, particularly in mammalian systems. As yet no conditions have been described for preparing plant DNA that is of sufficient length to allow similar long-range restriction mapping experiments in plant systems. In this report, we describe the application of the Schwartz and Cantor technique for preparing high molecular weight DNA from embedded tomato leaf protoplasts, as well as conditions for generating and fractionating large restriction fragments, by field inversion gel electrophoresis (FIGE). The bulk of DNA released from lysed protoplasts was at least 2 Mb in size and amenable to restriction digestion as shown by hybridizing Southern blots with, among others, a probe for the Adh-2 gene of tomato. Restriction fragments as large as 700 kb were detected. Chloroplast DNA is isolated intact, amenable to restriction analysis and, in its native form, not mobile in FIGE.     

Inflammation-induced DNA damage and damage-induced inflammation: a vicious cycle

Inflammation is the ultimate response to the constant challenges of the immune system by microbes, irritants or injury. The inflammatory cascade initiates with the recognition of microorganism-derived pathogen associated molecular patterns (PAMPs) and host cell-derived damage associated molecular patterns (DAMPs) by the pattern recognition receptors (PRRs). DNA as a molecular PAMP or DAMP is sensed directly or via specific binding proteins to instigate pro-inflammatory response. Some of these DNA binding proteins also participate in canonical DNA repair pathways and recognise damaged DNA to initiate DNA damage response. In this review we aim to capture the essence of the complex interplay between DNA damage response and the pro-inflammatory signalling through representative examples.     

Preparation of HMW DNA from Plant Nuclei and Chromosomes Isolated from Root Tips

Simple, fast and cost-effective method for preparation of DNA with high molecular weight (HMW DNA) from plant nuclei and mitotic chromosomes has been developed. The technique involves mechanical homogenization of formaldehyde-fixed root tips, purification of nuclei and/or chromosomes on sucrose gradient, embedding in low-melting-point agarose, and DNA isolation in agarose plugs. Alternatively, nuclei and chromosomes may be purified using flow cytometry. Majority of DNA obtained is megabase-sized and well digestible by restriction endonucleases. The method is highly efficient as microgram amounts of DNA can be obtained from only several milligrams of plant tissue. Handling negligible amounts of plant material reduces the consumption of chemicals. Furthermore, the use of root tips makes it possible to obtain high-quality DNA even from plant species with leaves that are rigid or rich in secondary metabolites such as polyphenols. It is expected that preparation of HMW DNA from root tip nuclei will facilitate long-range mapping and construction of large-insert DNA libraries also in these species. Successful isolation of HMW DNA from flow-sorted chromosomes opens a way for construction of chromosome-specific large-insert libraries in plants. This revised version was published online in July 2006 with corrections to the Cover Date.