Hydrogen peroxide is responsible for UVA-induced DNA damage measured by alkaline comet assay in HaCaT keratinocytes

We investigated the role of different reactive oxygen species (ROS) in ultraviolet A (UVA)-induced DNA damage in a human keratinocyte cell line, HaCaT. UVA irradiation increased the intracellular levels of hydrogen peroxide (H2O2), detected by a fluorescent probe carboxydichlorodihydrofluorescein, and caused oxidative DNA damage, single strand-breaks and alkali-labile sites, measured by alkaline single cell gel electrophoresis (comet assay). Superoxide anion (O2*-) was a likely substrate for H2O2 production since diethyldithiocarbamate (DDC), a superoxide dismutase blocker, decreased the level of intracellular H2O2. Hydrogen peroxide was shown to play a central role in DNA damage. Increasing the intracellular levels of H2O2 with aminotriazole (AT) (a catalase blocker) and buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis) potentiated the UVA-induced DNA damage. Exogenous H2O2 was also able to induce DNA damage. Since H2O2 alone is not able to damage DNA directly, we investigated the significance of the H2O2-derived hydroxyl radical (*OH). Addition of FeSO4, that stimulates *OH formation from H2O2 (Fenton reaction) resulted in a twofold increase of DNA-damage. Desferrioxamine, an iron chelator that blocks the Fenton reaction, prevented UVA-induced DNA damage. We also employed a panel of less specific antioxidants and enzyme modulators. Sodium selenite (Na-Se) present in glutathione peroxidase and thioredoxin reductase and addition of glutathione (GSH) prevented DNA-damage. Tocopherol potently prevented UVA-and H2O2-induced DNA damage and reduced intracellular H2O2 -levels. Ascorbic acid reduced H2O2 production, but only partly prevented DNA damage. Singlet oxygen (1O2) did not seem to play an important role in the UVA-induced DNA-damage since the specific 1O2 scavenger sodium azide (NaN3) and the less specific 1O2 scavenger beta-carotene did not markedly prevent either DNA-damage or H2O2 production. In conclusion the conversion of H2O2 to *OH appears to be the most important step in UVA-induced generation of strand breaks and alkali-labile sites and the bulk H2O2 appears to originate from O2*- generated by UVA irradiation.     

Assessment of gamma ray-induced DNA damage in Lasioderma serricorne using the comet assay

We attempted a DNA comet assay under alkaline conditions to verify the irradiation treatment of pests. Lasioderma serricorne (Fabricius) were chosen as test insects and irradiated with gamma rays from a ⁶⁰Co source at 1 kGy. We conducted the comet assay immediately after irradiation and over time for 7 day. Severe DNA fragmentation in L. serricorne cells was observed just after irradiation and the damage was repaired during the post-irradiation period in a time-dependent manner. The parameters of the comet image analysis were calculated, and the degree of DNA damage and repair were evaluated. Values for the Ratio (a percentage determined by fluorescence in the damaged area to overall luminance, including intact DNA and the damaged area of a comet image) of individual cells showed that no cells in the irradiated group were included in the Ratio<0.1 category, the lowest grade. This finding was observed consistently throughout the 7-day post-irradiation period. We suggest that the Ratio values of individual cells can be used as an index of irradiation history and conclude that the DNA comet assay under alkaline conditions, combined with comet image analysis, can be used to identify irradiation history.     

高效液相色谱-电感耦合等离子体质谱研究Medaka体内水溶性汞结合蛋白

采用高效液相色谱与电感耦合等离子体质谱联用技术,对不同形态汞暴露后的Medaka肝脏和脑组织中水溶性蛋白进行研究,以体积排阻柱为液相分离柱,流动相为0.15 mol/L NH4Ac,流速为0.5 mL/min.利用电感耦合等离子体质谱对Medaka肝脏和脑组织提取液中铜、锌和汞的含量进行了分析,检出限为0.01～0.0...     

Single-cell gel/comet assay applied to the analysis of UV radiation-induced DNA damage in Rhodomonas sp. (Cryptophyta).

The single-cell gel/comet assay is an electrophoretic technique used to detect single-strand breaks in DNA. Damage is assessed examining individual cells under an epifluorescent microscope. UV-induced DNA damage consists mostly of the formation of pyrimidine dimers; therefore, most of the damage cannot be detected using a standard comet assay. The enzyme T4 endonuclease V breaks DNA strands at sites of pyrimidine dimers. The main objective of this work is to evaluate the comet assay to detect UV-induced damage in DNA after an initial treatment of cells with T4 endonuclease V. This work was conducted on Rhodomonas sp. (Cryptophyta), a marine unicellular flagellate. Cells of Rhodomonas sp. were exposed to 12 h visible + ultraviolet-A + ultraviolet-B (VIS + UVA + UVB) and VIS (control), with and without T4 endonuclease V. Cells exposed to VIS + UVA + UVB showed approximately 200% more damage than control if these were treated with T4 endonuclease V. Rhodomonas sp. were exposed to 3, 6, 9 and 12 h of VIS, VIS + UVA and VIS + UVA + UVB. Damage induced by VIS + UVA + UVB as detected by the comet assay increased along with exposure time. However, damage caused by VIS and VIS + UVA remained relatively constant at all times. Results of this study indicate that the comet assay is more sensitive to UV radiation damage when used in conjunction with T4 endonuclease V. This modification of the comet assay can be used as an alternative technique to detect DNA damage in single cells caused by UV radiation.     

Oryzias latipes鱼体对不同形态镧的积累特征

在试验室条件下研究了Oryzias latipes鱼体对不同形态轻稀土La的积累特征及它对游离La吸收的动力学过程,并初步探讨了鱼对La的吸收机制。结果表明,试验鱼对La的积累量随水相游离La浓度增高、暴露时间增长而增加;水相富里酸的存在明显降低了La对鱼对生物有效性。此外,试验鱼也可以通过摄食获取水相中的La。     

Validation of raw data measurements in the comet assay

General guidance recently proposed for the comet assay concluded that “the method should be adjusted scientifically at each laboratory to obtain valid and reproducible results”. However, the comet widely used metrics, Tail DNA and Tail moment, are actually based on a ratio of fluorescence signals, a relative and semi-quantitative measurement, and are quite difficult to validate according to classical criteria. As the validation of analytical methods increasingly becomes an absolute requirement in many fields, this paper investigates a scheme to study the variability of raw data measurements for computer-assisted comet measurement, including the between-operators reproducibility. In the overall analysis process, we show that the image acquisition step gives the highest variability, notably for the Tail length parameter that negatively influences the Olive tail moment. However, when the operator interacts with the system to correct obviously mistaken measurements, the reproducibility is sensibly improved. For the metrics Tail DNA and Olive tail moment, the total variability in measurements for a panel of comets quantified by different operators in real conditions is about 4%. The proposed validation scheme allows to assess the measurement process and to verify if there are any major difference between trained operators, an essential requirement for long-term investigations.     

Detection of Phakopsora pachyrhizi by polymerase chain reaction (PCR) and use of germination test and DNA comet assay after e-beam processing in soybean

Soybean harvest is the main agribusiness in Brazil, which is the second largest exporter in the world and has a revenue of billions of dollars. Asian dust is caused by the fungus Phakopsora pachyrhizi and its dissemination is difficult to control, since it occurs through wind dispersion. Actually P. pachyrhizi is found in different parts of the world. Electron beam treatment could be an alternative process to minimize these losses, especially for the grains exportation industry. Besides the possibility of being disconnected when not in use, this source does not need to be reloaded, is easily available and, streamlines the process and reduces logistics costs. The present work aims to identify, by the polymerase chain reaction technique (PCR), the P. pachyrhizi fungus presence in the irradiated soybeans and the possibility to use radiation treatment as a sanitary alternative. Doses 0, 1.0, 2.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 kGy (IPEN-CNEN/SP Electron Accelerator) were applied and two fast-screening methods were performed: DNA comet assay (for the detection of DNA damage) and germination test (for the measurement of roots inhibition). These tests are very easy to carry out and measure damage response depending on radiation dose.     

Detection and mechanistic investigation of halogenated benzoquinone induced DNA damage by photoelectrochemical DNA sensor

Halogenated phenols are widely used as biocides and are considered to be possibly carcinogenic to humans. In this report, a previously developed photoelectrochemical DNA sensor was employed to investigate DNA damage induced by tetra-halogenated quinones, the in vivo metabolites of halogenated phenols. The sensor surface was composed of a double-stranded DNA film assembled on a SnO₂ semiconductor electrode. A DNA intercalator, Ru(bpy)₂(dppz)²⁺, was allowed to bind to the DNA film and produce photocurrent upon light irradiation. After the DNA film was exposed to 300 μM tetrafluoro-1,4-benzoquinone (TFBQ), the photocurrent dropped by 20%. In a mixture of 300 μM TFBQ and 2 mM H₂O₂, the signal dropped by 40%. The signal reduction indicates less binding of Ru(bpy)₂(dppz)²⁺ due to structural damage of ds-DNA in the film. Similar results were obtained with tetra-1,4-chlorobenzoquinone (TCBQ), although the signal was not reduced as much as TFBQ. Fluorescence measurement showed that TFBQ/H₂O₂ generated more hydroxyl radicals than TCBQ/H₂O₂. Gel electrophoresis proved that the two benzoquinones produced DNA strand breaks together with H₂O₂, but not by themselves. Using the photoelectrochemical sensor, it was also found that TCBQ covalently bound with DNA did not produce additional oxidative damage in the presence of H₂O₂. The combined photoelectrochemistry, gel electrophoresis, and fluorescence data revealed distinctive differences between TFBQ and TCBQ in terms of DNA adduct formation and hydroxyl radical generation.     

DNA comet assay: A simple screening technique for identification of some irradiated foods

DNA Comet Assay method was carried out to detect irradiation treatment of some foods like meat, spices, beans and lentils. The fresh meat of cow and duck were irradiated up to radiation doses of 3 kGy, the spices (cardamoms and cumin black) were irradiated to radiation doses of 5, 10, 15 and 20 kGy while the beans (black beans and white beans) and lentils (red and green lentils) were irradiated to 0.5 and 1 kGy. All the foods were then analyzed for radiation treatment using simple microgel electrophoresis of single cells or nuclei (DNA Comet Assay). Sedimentation, lysis and staining times were adjusted to get optimized conditions for correct and easy analysis of each food. Using these optimized conditions, it was found out that radiation damaged DNA showed comets in case of irradiated food samples, whereas in non-treated food samples, round or conical spots of stained DNA were visible. Shape, length and intensity of these comets were also radiation dose dependent. Screening of unirradiated and irradiated samples by Comet Assay was successful in the case of all the foods under consideration under the optimized conditions of assay. Therefore, for different kinds of irradiated foods studied in the present study, the DNA Comet Assay can be used as a rapid, simple and inexpensive screening test.     

Detection of irradiation treatment of foods using DNA ‘comet assay’

Microgel electrophoresis of single cells (DNA comet assay) has been investigated to detect irradiation treatment of some food samples. These samples of fresh and frozen rainbow trout, red lentil, gram and sliced almonds were irradiated to 1 or 2 kGy using 10 MeV electron beam from a linear accelerator. Rainbow trout samples yielded good results with samples irradiated to 1 or 2 kGy showing fragmentation of DNA and, therefore, longer comets with no intact cells. Unirradiated samples showed shorter comets with a significant number of intact cells. For rainbow trout stored in a freezer for 11 days the irradiated samples can still be discerned by electrophoresis from unirradiated samples, however, the unirradiated trouts also showed some longer comets besides some intact cells. Radiation treatment of red lentils can also be detected by this method, i.e. no intact cells in 1 or 2 kGy irradiated samples and shorter comets and some intact cells in unirradiated samples. However, the results for gram and sliced almond samples were not satisfactory since some intact DNA cells were observed in irradiated samples as well. Probably, incomplete lysis has led to these deviating results.     

Comparison of comet assay parameters for estimation of genotoxicity by sum of ranking differences

The genotoxic potential of waters in six rivers and reservoirs from Serbia was monitored in different tissues of chub (Squalius cephalus L. 1758) with the alkaline comet assay. The comet assay, or single-cell gel electrophoresis, has a wide application as a simple and sensitive method for evaluating DNA damage in fish exposed to various xenobiotics in the aquatic environment. Three types of cells, erythrocytes, gill cells, and liver cells, were used for assessing DNA damage. Images of randomly selected cells were analyzed with a Leica fluorescence microscope and image analysis by software (Comet Assay IV Image analysis system, PI, UK). Three parameters (tail length—l, tail intensity—i, and Olive tail moment—m) were analyzed on 1,700 nuclei per cell type. The procedure for sum of ranking differences (SRD) was implemented to compare different types of cells and different parameters for estimation of DNA damage. Regarding our nine different estimations of genotoxicity: tail length, intensity, and moment in erythrocytes (rel, rei, rem), liver cells (rll, rli, rlm), and gill cells (rgl, rgi, rgm), the SRD procedure has shown that the Olive tail moment and tail intensity are (almost) equally good parameters; the SRD value was lower for the tail moment and tail intensity than for tail length in the case of all types of cells. The least reliable parameter was rel; close to the borderline case were rei, rll, and rgl (～5 % probability of random ranking).

Detection of DNA damage by exploiting the distance dependence of the electrochemiluminescence energy transfer between quantum dots and gold nanoparticles

Microchimica Acta - We have developed a platform to detect DNA damage induced by perfluorooctanoic acid (PFOA) by measuring the electrochemiluminescence (ECL) of a layer-by-layer electrostatic...     

Detection of a urinary biomaker for oxidative DNA damage 8-hydroxydeoxyguanosine by capillary electrophoresis with electrochemical detection

8-Hydroxydeoxyguanosine (8-OHdG) is present in urine as a result of oxidative DNA damage associated with age-related diseases such as cancer. In this report a method is presented for the detection of 8-OHdG in human morning urine utilizing capillary electrophoresis with electrochemical detection (CEEC). The limit of detection for a aqueous standard of 8-OHdG is 50 nM (signal to noise ratio S/N = 3). A single solid-phase extraction (SPE) step with a C18 column is used for sample cleanup and 20-fold preconcentration of the urine before analysis by CEEC. Optimized conditions for analysis of extracted urine are E(app) = 0.5 V vs. Ag/AgCl with 20 mM sodium borate/20% MeOH v/v, pH 9, as the background electrolyte, and a separation voltage of 22 kV. The concentration of 8-OHdG varied from 6 to 86 nM with an average value of 42 +/- 26.9 nM for four healthy female and four healthy male subjects between the ages of 23 and 43.     

Detection of telomere damage as a result of strand breaks in telomeric and subtelomeric DNA

Telomeres are the tandem repetitive DNA sequences at both ends of a chromosome with a repeating unit of TTAGGG. The integrity of a telomere is crucial to chromosomal stability and cellular viability. Damages to telomere DNA disrupt telomere integrity and accelerate telomere shortening. We describe a method for the assessment of strand breaks in the telomere/subtelomere region in cultured cells. Cells were embedded in agarose plugs and subjected to lysis and alkaline treatment to relax the DNA double helix. The telomere fragments as the result of strand breaks in the telomere/subtelomere region were then separated from the genomic DNA by electrophoresis, blotted onto membranes, and detected by a probe specific to the telomere sequence. Because of the large content of the telomere in human cells and the fact that telomere DNA is much more prone to damage than the bulk genomic DNA, the analysis may serve as a good indication of general DNA damage as well.     

Detection of damage to DNA and antioxidative activity of yeast polysaccharides at the DNA-modified screen-printed electrode

A simple procedure for the voltammetric detection of the DNA damage and antioxidants protecting DNA from its damage using a disposable electrochemical DNA biosensor is reported. The carbon-based screen-printed electrode (SPE) modified by a surface layer of the calf thymus double stranded (ds) DNA was used as a working electrode in combination with a silver/silver chloride reference electrode and a separate platinum auxiliary electrode. The [Co(phen)(3)](3+) ion served as the dsDNA redox marker and the [Cu(phen)(2)](2+) and [Fe(EDTA)](-) complex compounds were used as the DNA cleavage agents under the reduction by a chemical reductant (ascorbic acid). Four yeast polysaccharides with different chemical structure were investigated as the antioxidants within the concentration range of 0.05-4 mg ml(-1) in the cleavage mixture. A remarkable antioxidative activity of polysaccharides in order mannan (Candida krusei)>extracellular glucomannan (Candida utilis)>mannan (Candida albicans)>glucomannan (C. utilis) was found which is in agreement with that refered to trolox (a structural derivative of alpha-tocopherol) and determined by photochemiluminescent method.     

Metal concentrations in Japanese medaka,mosquitofish and insect larvae living in uncontaminated rivers in Kumamoto,Japan

Elemental concentrations in Japanese medaka and mosquitofish collected from uncontaminated rivers in Kumamoto, Japan were analyzed by instrumental neutron activation analysis with k ₀ standardization method to know the background levels. A statistical analysis indicated the difference in metal concentrations among rivers and species. Background levels of elemental concentrations in some aquatic insect larvae were analyzed and metal concentrations were different among species, though they were collected at the same point. Enrichment factors of heavy metals in insect larvae were in the order of 10³−10⁴ suggesting effectiveness of insect larvae as bioindicator.     

Modulation of photo-oxidative DNA damage by cationic surfactant complexation

The natural packaging of DNA in the cell by histones provides a particular environment affecting its sensitivity to oxidative damage. In this work, we used the complexation of DNA by cationic surfactants to modulate the conformation, the dynamics, and the environment of the double helix. Photo-oxidative damage initiated by benzophenone as the photosensitizer on a plasmid DNA complexed by dodecyltrimethylammonium chloride (DTAC), tetradecyltrimethylammonium chloride (TTAC), cetyltrimethyammonium chloride (CTAC) and bromide (CTAB) was detected by agarose gel electrophoresis. By fluorescent titration in the presence of ethidium bromide (EB) and agarose gel electrophoresis, we experimentally confirmed the complexation diagrams with a critical aggregation concentration on DNA matrix (CAC DNA) delimiting two regions of complexation, according to the DNA-phosphate concentration. The study of the photo-oxidative damage shows, for the first time, a direct correlation between the DNA complexation by these surfactants and the efficiency of DNA cleavage, with a maximum corresponding to the CAC DNA for DTAC and CTAC, and to DNA neutralization for CTAC and CTAB. The localization of a photosensitizer having low water solubility, such as benzophenone, inside the hydrophobic domains formed by the surfactant aggregated on DNA, locally increases the photoinduced cleavage by the free radical oxygen species generated. The inefficiency of a water-soluble quencher of hydroxyl radicals, such as mannitol, confirmed this phenomenon. The detection of photo-oxidative damage constitutes a new tool for investigating DNA complexation by cationic surfactants. Moreover, highlighting the drastically increased sensitivity of a complexed DNA to photo-oxidative damage is of crucial importance for the biological use of surfactants as nonviral gene delivery systems.     

A selective, noncovalent assay for base flipping in DNA

Base flipping, the conformational change of a nucleobase to an extrahelical position, is a key step in the enzymatic repair of damaged DNA. An assay that can detect the flipped-out species in free solution without covalent modification of the DNA would be desirable. The design and synthesis of a simple, sensitive, and rapid assay using specific noncovalent binding to pyrimidines by zinc-cyclen and a commonly used fluorescent reporter group, dansyl, is reported. The binding of the zinc-cyclen unit to a flipped-out thymine base results in a change in the fluorescent properties of the dansyl group that is distinct from nonspecific binding to duplex DNA or intercalation into either the flipped-in or flipped-out species. The assay was tested using fluorescence spectroscopy and detection at 533 +/- 5 nm with normal and abasic duplex DNA as negative and positive controls. The data obtained are fitted to a one-site binding model to determine the equilibrium constant for the two-step process involving base flipping and binding to be approximately 10-6 M.     

X-ray induced damage in DNA monitored by X-ray photoelectron spectroscopy

In this work, the chemical changes in calf thymus DNA samples were analyzed by X-ray photoelectron spectroscopy (XPS). The DNA samples were irradiated for over 5 h and spectra were taken repeatedly every 30 min. In this approach the X-ray beam both damages and probes the samples. In most cases, XPS spectra have complex shapes due to contributions of C, N, and O atoms bonded at several different sites. We show that from a comparative analysis of the modification in XPS line shapes of the C 1s, O 1s, N 1s, and P 2p peaks, one can gain insight into a number of reaction pathways leading to radiation damage to DNA.     

N-acetyl-L-cysteine prevents DNA damage induced by UVA, UVB and visible radiation in human fibroblasts

The thiol N-acetyl-L-cysteine (NAC) is a source of cysteine for the synthesis of the endogenous antioxidant glutathione (GSH) which is depleted by ultraviolet radiation. It is also associated with the scavenging of reactive oxygen species (ROS). In this study the effects of NAC were examined in cultured human fibroblasts during prolonged exposure to ultraviolet B (UVB), ultraviolet A (UVA) and visible irradiation (280-700 nm), delivered by a 150 W xenon-arc lamp. The alkaline comet assay was used to assess the DNA damage in individual cells. It was found that incubating skin and lung fibroblasts at 37 degrees C for 1 h with an optimal 6 mM NAC supplement prior to light exposure, significantly reduced the level of DNA damage in both cell types, however, the skin fibroblasts were less sensitive to xenon-arc lamp irradiation than lung fibroblasts. NAC incubation resulted in an initial delay in DNA damage when the cells were irradiated. There was also a significant reduction in the overall levels of DNA damage observed with continued irradiation. NAC significantly reduced the DNA damage produced in lung fibroblasts depleted of normal GSH protection by the glutamylcysteinyl synthetase inhibitor, L-buthionine-[S,R]-sulfoximine. Although the specific mechanism of NAC protection has not yet been elucidated, these results support the hypothesis that NAC may protect the cells directly, by scavenging ROS induced by UVA and visible radiation, and indirectly by donating cysteine for GSH synthesis.