Effects of ethidium bromide on DNA loop organisation in human lymphocytes measured by anomalous viscosity time dependence and single cell gel electrophoresis.

The effects of ethidium bromide (EtBr) on human lymphocytes were studied by the method of anomalous viscosity time dependence (AVTD) and by the comet assay. EtBr at low concentrations increased the maximum viscosity and time of radial migration as measured with AVTD at neutral conditions of lysis. A pronounced relaxation of DNA loops was observed with the neutral comet assay. The maximal comet length corresponded to 2 Mb DNA loops. At high concentrations of EtBr, 2 mg/ml, significant reduction in AVTD below control level was seen that suggested hypercondensation of chromatin. The hypercondensation was directly observed with the neutral comet assay. EtBr did not induce DNA strand breaks as measured by the alkaline comet assay. The hypercondensed nuclei could be decondensed by irradiation with gamma-rays or exposure to light. The data provide evidence that EtBr at high concentrations resulted in hypercondensation of chromatin below control level. The comet assay confirmed that the increase in AVTD peaks deals with relaxation of loops and AVTD decrease is caused by chromatin condensation. The prediction of the AVTD theory for a correlation between time of radial migration and condensation of chromatin was verified. Further, the data show that the comet assay at neutral conditions of lysis is rather sensitive to DNA loop relaxation in the absence of DNA damage. Finally, donor specificity was found for the hypercondensation.     

Evaluation of the nuclear DNA Diffusion Assay to detect apoptosis and necrosis

We applied the nuclear DNA Diffusion Assay, described as an accurate tool to estimate apoptotic and necrotic cells [N.P. Singh, A simple method for accurate estimation of apoptotic cells, Exp. Cell Res. 256 (2000) 328-337] to tobacco root and leaf cells. In this assay, isolated nuclei are embedded in an agarose microgel on a microscope slide and low molecular-weight DNA fragments diffuse into the microgel. Exposure of the roots to hydrogen peroxide significantly increased the average nuclear area of isolated nuclei. After 4 and 24 h of recovery, all DNA damage was repaired. The data clearly demonstrate that the manifestation of diffused nuclei upon exposure to hydrogen peroxide is not the result of non-repairable apoptotic or necrotic DNA fragmentation, but represents repairable genotoxin-induced DNA damage. In contrast, treatment with the alkylating agent ethyl methanesulphonate (EMS) followed by 24 h of recovery produced a significant increase in the average nuclear area. The contribution of apoptosis to this increase cannot be excluded. Heat treatment of leaves at 50 degrees C for 1-15 min leading to necrosis, and treatment of isolated nuclei with DNase-I, which digests DNA to nucleosome-sized fragments as during apoptosis, also led to a dose-dependent increase in the nuclear area. The use of different fluorochromes (ethidium bromide, DAPI or YOYO-1) for DNA staining yielded similar results in the DNA Diffusion Assay. As all types and sizes of diffused nuclei were observed after EMS and hydrogen peroxide treatments, we were unable to differentiate, on the basis of the structure of the nuclei, between apoptotic or necrotic DNA fragmentation and other types of genotoxin-induced DNA damage in plants.     

Analysis of chromate-induced DNA-protein crosslinks with the comet assay

Modifications of the comet assay have been introduced to measure crosslinks by determining the reduction of induced DNA migration. Our previous results indicated that the modified protocol of the alkaline comet assay is a sensitive tool for the detection of formaldehyde-induced DNA-protein crosslinks. But results for mitomycin C and cisplatin suggested that the modified protocol is not well suited for the evaluation of DNA-DNA crosslinkers. We now used the comet assay to investigate in V79 cells the effect of potassium chromate (K(2)CrO(4)), another DNA-protein crosslinker, to see whether the results obtained for formaldehyde can be generalized. However, chromate did not reduce spontaneous or radiation-induced DNA migration in the alkaline (pH 13) comet assay but led to a small but significant induction of DNA migration. A crosslinking effect of chromate could also not be detected with the alkaline comet assay after postincubation of cells in normal medium after chromate treatment to enable repair of other (migration-inducing) lesions that might mask the crosslinking effect. Exposure of slides to proteinase K further increased DNA migration of chromate-treated cells, thus indicating the presence of DNA-protein crosslinks. In contrast to the alkaline comet assay, a "neutral" version at pH 9 was suited to demonstrate reduced induction of DNA migration after gamma-irradiation of chromate-treated cells. The crosslinking effect was seen immediately at the end of the chromate treatment as well as after a 3h postincubation period. Using the "neutral" protocol in combination with proteinase K, we were able to demonstrate the presence of DNA-protein crosslinks as the probable cause for the migration-reducing effect. Further investigations will have to show whether this protocol can be recommended as a universal approach for the detection of DNA-protein crosslinks and also of DNA-DNA crosslinks with the comet assay.     

Selective staining by 4'', 6-diamidine-2-phenylindole of nanogram quantities of DNA in the presence of RNA on gels.

4', 6-Diamidine-2-phenylindole.2HCl (DAPI) forms fluorescent complexes with double-stranded (ds) DNA but not with ds RNA as shown by fluorescence titration. The widely used dye ethidium bromide (EB) forms fluorescent complexes with both types of nucleic acids. Also, in contrast to EB, DAPI forms much weaker fluorescent complexes with single-stranded DNA than with ds DNA. These observations were utilized to develop staining procedures for the selective visualization of ds DNA on gels. The use of DAPI in addition to EB for staining makes possible the localization of ds DNA and other species of nucleic acids on a single gel.     

Application of the novel nucleic acid dyes YOYO-1, YO-PRO-1, and PicoGreen for flow cytometric analysis of marine prokaryotes.

Novel blue light-excited fluorescent dyes for nucleic acids (YOYO-1, YO-PRO-1, and PicoGreen) were tested on cultures of Escherichia coli and of a variety of marine prokaryotes. Results of flow cytometric DNA analyses were compared with those obtained with the UV-excited dyes bis-benzimide Hoechst 33342 or 4', 6-diamidino-2-phenylindole (DAPI). YOYO-1, YO-PRO-1, and PicoGreen can be used only on aldehyde-fixed cells and need to be supplemented with cofactors such as potassium, citrate, or EDTA. They are highly sensitive to ionic strength. Consequently, seawater culture samples cannot be stained directly with these dyes and require at least a 10-fold dilution with distilled water to obtain reliable fluorescence signals. After treatment with RNase, coefficients of variation for the G1 peak of the DNA distributions of the different strains tested with YOYO-1 or PicoGreen indicated in general an improvement over Hoechst 33342 staining. These novel dyes can be used to enumerate prokaryotic cells by flow cytometry, as demonstrated with E. coli. However, their sensitivity to ionic strength makes them unsuitable for cell cycle analysis in natural samples.     

A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands.

We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids.     

Different effects of 33258 Hoechst and DAPI in fluorescent staining of sister chromatids differentially substituted with bromodeoxyuridine

Summary After substitution with 5-bromodeoxyuridine (BrdUrd) for two rounds of replication, chromosomes in cytological preparations stained with 33258 Hoechst show upon epiluminescence an immediate differential sister chromatid fluorescence. When stained with DAPI, however, which has a structural resemblance to part of the 33258 Hoechst molecule, such a differential pattern of fluorescence was only induced after some delay. Upon restaining with the same dye the differential fluorescence appeared instantly. In preparations double stained with ethidium bromide and 33258 Hoechst the induction of a differential staining of sister chromatids with 33258 Hoechst was not accompanied by a differential staining with ethidium bromide. Once a differential staining was obtained with DAPI in preparations double stained with ethidium bromide and DAPI, the ethidium bromide pattern also appeared to be differential upon subsequent observation. No differentiation could be obtained with ethidium bromide alone. The observations described in the case of 33258 Hoechst staining are in agreement with a molecular quenching by BrdUrd without gross structural consequences for the DNA. In the case of DAPI staining, however, there occurs a differential photolysis of BrdUrd-substituted DNA. Besides the nature, most likely the size, of the fluorochrome molecules themselves, the state of the fixed chromatin appeared also to play a role in determining the mechanism of the sister chromatid differentiation: after prolonged incubation in buffer, BrdUrd-containing chromosomes stained with 33258 Hoechst showed a differential staining evidently caused by photolysis, indicating that they had become more susceptible to light.     

Application of the comet assay in erythrocytes of Oreochromis niloticus (Pisces): A methodological comparison

The present study applied the comet assay to erythrocytes of Oreochromis niloticus with the aim of improving protocols to detect DNA damage in these cells, by using two distinct pHs (pH = 12.1 and pH > 13) and evaluating whether there is a correspondence between silver and ethidium bromide staining. Comets were visually examined and, the frequency of cells with and without damage was obtained, as well as the distribution of classes and scores. By using the Kruskal-Wallis test, our results revealed that pH 12.1 is more effective, although both pHs can be used. Our findings also suggest that silver staining can substitute ethidium bromide, an expensive and highly toxic stain that requires specific equipment for examination.     

Assessment of the distribution of nucleic acid intercalators in yeast cells by the pseudospectral image analysis

The intracellular location of nucleic acid intercalators (NAI) in live (not fixed) Saccharomyces cerevisiae cells has been studied using fluorescence microscopy combined with computer pseudospectral image analysis. Three NAI: the anthracycline anticancer drug doxorubicin and the nucleic acid dyes ethidium bromide (E) and 4',6-diamidino-2-phenylindole (DAPI) were used. All three NAI were shown to be localized in nuclei and mitochondria. In contrast to DAPI, which interacted only with DNA, a large fraction of doxorubicin and ethidium bromide apparently bound to mitochondrial membranes. Upon combined application, a competition between these intercalators for binding sites in the nuclear and mitochondrial DNA occurred. It was concluded that this approach may be used in designing new DNA-targeted drugs and in preliminary studies of their interaction with eukaryotic cells.     

An ethidium bromide-agarose plate assay for the nonradioactive detection of cDNA synthesis

Ethidium bromide was used to determine the success of cDNA synthesis reactions. Since ethidium bromide in agarose can be used to quantitate RNA and DNA, conditions under which the greater fluorescence of double-stranded DNA (dsDNA) is utilized were devised to assay dsDNA synthesis from mRNA. Ethidium bromide at 5 micrograms/ml in agarose allowed quantitative detection of cDNA in the range of 0.03 to 0.0015 microgram. Sodium dodecyl sulfate had an adverse effect on the measurement of cDNA. Subsequent cDNA analysis by alkaline gel electrophoresis and staining in 5 micrograms/ml ethidium bromide allowed accurate and rapid sizing of cDNA and required only 0.1-0.05 microgram cDNA.     

Comparison of ethidium bromide and 4′,6-diamidino-2-phenylindole as quantitative fluorescent stains for DNA in agarose gels

Two methods of quantitative, fluorescent detection of DNA bands in agarose gels separated by electrophoresis were evaluated for sensitivity and linearity of response. Comparisons of ethidium bromide staining with a method using 4′,6-diamidino-2-phenylindole (DAPI) developed in this work showed that DAPI is several times more sensitive than ethidium bromide in conditions of comparable background flourescence. Optimum flourescent staining and detection conditions for DNA bands in agarose gels using DAPI are desctribed, and advantages of this method over other fluorescent detection methods are discussed.     

Assessment of the distribution of nucleic acid intercalators in yeast cells by pseudospectral image analysis

The intracellular location of nucleic acid intercalators (NAI) in native (not fixed) Saccharomyces cerevisiae cells has been studied using fluorescence microscopy combined with computer pseudospectral image analysis. Three NAI: anthracycline anticancer drug doxorubicin and nucleic acid dyes ethidium bromide and 4′,6-diamidino-2-phenylindole (DAPI) were used. All three NAI were shown to be localized in nuclei and mitochondria. In contrast to DAPI, which interacted only with DNA, a large fraction of doxorubicin and ethidium bromide apparently bound to mitochondrial membranes. Upon combined application, competition between these intercalators for binding sites in the nuclear and mitochondrial DNA occurred. It was concluded that this approach may be used in designing new DNA-targeted drugs and in preliminary studies of their interaction with eukaryotic cells.     

Alkaline, Endo III and FPG modified comet assay as biomarkers for the detection of oxidative DNA damage in rats with experimentally induced diabetes

Increased production of reactive oxygen species under diabetic condition underlines the higher oxidatively damaged DNA in different tissues. However, it is practically difficult to assess the oxidatively damaged DNA in different internal organs. Therefore, the present study was aimed to evaluate the extent of oxidative stress-induced DNA damage in different organs with the progression of diabetes. Diabetic and control Sprague Dawley rats were sacrificed in time-dependent manner and the lung, liver, heart, aorta, kidney, pancreas and peripheral blood lymphocytes (PBL) were analyzed for both alkaline and modified comet assay with endonuclease-III (Endo III) and formamidopyrimidine-DNA glycosylase (FPG) (hereafter called modified comet assay) for the detection of oxidative DNA damage. The statistically significant increase in olive tail moment (OTM) was found in all the tested tissues. The extent of DNA damage was increased with the progression of diabetes as revealed by the parameter of OTM in alkaline and modified comet assay. Further, the positive correlations were observed between OTM of the lung, liver, heart, aorta, kidney and pancreas with PBL of diabetic rat in the alkaline and modified comet assay. Moreover, significant increase in the 8-oxodG positive nuclei in the lung, liver, heart, aorta, kidney and pancreas was observed in 4th and 8th week diabetic rat as compared to control. Results of the present study clearly indicated the suitability of alkaline and modified comet assay for the detection of multi-organ oxidative DNA damage in streptozotocin (STZ)-induced diabetic rat and showed that damaged DNA of PBL can be used as a suitable biomarker to assess the internal organs response to DNA damage in diabetes.     

Genotoxicity and mutagenicity of iron and copper in mice

The toxicity of trace metals is still incompletely understood. We have previously shown that a single oral dose of iron or copper induces genotoxic effects in mice in vivo, as detected by single cell gel electrophoresis (comet assay). Here, we report the effect of these metals on subchronic exposure. Mice were gavaged for six consecutive days with either water, 33.2 mg/kg iron, or 8.5 mg/kg copper. On the 7th day, the neutral and alkaline comet assays in whole blood and the bone marrow micronucleus (MN) test were used as genotoxicity and mutagenicity endpoints, respectively. Particle induced X-ray emission was used to determine liver levels of the metals. Females showed a slightly lower DNA damage background, but there was no significant difference between genders for any endpoint. Iron and copper were genotoxic and mutagenic. While copper was more genotoxic in the neutral version, iron was more genotoxic in the alkaline version of the comet assay. Copper induced the highest mutagenicity as evaluated by the MN test. Iron was not mutagenic to male mice. Iron is thought to induce more oxidative lesions than copper, which are primarily detected in the alkaline comet assay. Treatment with iron, but not with copper, induced a significant increase in the hepatic level of the respective metal, reflecting different excretion strategies.     

Fluorometric determination of DNA and RNA in Chlamydomonas using ethidium bromide

By using the fluorescence enhancement of ethidium bromide bound to nuclei acid, a very rapid, simple and sensitive assay of DNA in the green alga Chlamydomonas has been devised. Total fluorescence (DNA + RNA) was determined by complex formation with ethidium bromide in a cell lysate made by mixing cell samples with lauroyl sarcosinate, EDTA and NaOH and incubating the mixture for 5 min at room temperature followed by neutralization. For determination of DNA the RNA was digested by incubating the cell sample in te alkaline lysis solution for 45 min at 60 degrees C followed by neutralization, and complex formation with ethidium bromide. Quenching of the fluorescence due to cellular pigments was corrected for using an internal DNA standard.     

Molecular mechanisms and kinetics between DNA and DNA binding ligands

Mechanical properties of single double-stranded DNA (dsDNA) in the presence of different binding ligands were analyzed in optical-tweezers experiments with subpiconewton force resolution. The binding of ligands to DNA changes the overall mechanic response of the dsDNA molecule. This fundamental property can be used for discrimination and identification of different binding modes and, furthermore, may be relevant for various processes like nucleosome packing or applications like cancer therapy. We compared the effects of the minor groove binder distamycin-A, a major groove binding alpha-helical peptide, the intercalators ethidium bromide, YO-1, and daunomycin as well as the bisintercalator YOYO-1 on lambda-DNA. Binding of molecules to the minor and major groove of dsDNA induces distinct changes in the molecular elasticity compared to the free dsDNA detectable as a shift of the overstretching transition to higher forces. Intercalating molecules affect the molecular mechanics by a complete disappearance of the B-S transition and an associated increase in molecular contour length. Significant force hysteresis effects occurring during stretching/relaxation cycles with velocities >10 nm/s for YOYO-1 and >1000 nm/s for daunomycin. These indicate structural changes in the timescale of minutes for the YOYO-DNA and of seconds for the daunomycin-DNA complexes, respectively.     

Detection of excision repaired DNA damage in the comet assay by using Ara-C and hydroxyurea in three different cell types

Because of its characteristics, the comet assay has been used to evaluate the ability of virtually any type of eukaryotic cell to repair different kinds of DNA damage, including double and single strand breaks and base damage. The ability to detect excision repair sites using the alkaline version can be enhanced by the inclusion of repair inhibitors, DNA synthesis inhibitors, or chain terminators. In this sense, we evaluated the ability of hydroxyurea (HU) and cytosine arabinoside (Ara-C), for detecting lesions produced by the alkylating agents ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) in three different cell systems. Two hundred cells for experimental point were analyzed in the alkaline version of the comet assay, and the results are evidences of the utility of the assay to detect alkylation of bases in the cells lines MRC-5 and TK-6, as the treatment with HU +Ara-C significantly increases both the basal and induced frequency of DNA damage. The use of whole blood, although it detected the effects of MMS, with and without repair inhibitors, failed to detect the effect of the selected dose of EMS and does not permit detection increases in the background level.     

Genotoxicity of cadmium chloride in human lymphocytes evaluated by the comet assay and cytogenetic tests

Peripheral blood lymphocytes were tested in vitro for genotoxic effects of cadmium chloride. Whole blood samples of four healthy, non-smoking subjects were preincubated with CdCl2 in concentrations of 10(-4), 10(-3), and 5 . 10(-3) mol/L for three hours before the cells were assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay) or cultivated for chromosomal aberrations (CA), sister chromatid exchanges (SCE), and the micronucleus (MN) test. The comet assay showed notable interindividual differences. The results of the cytogenetic tests showed an increase in the frequency of CA, MN, and SCE with CdCl2 in the treated cultures, yet none was able to show a correlation between concentrations of cadmium chloride and the frequency of damages. The MN slides were stained with Giemsa and with DNA fluorochrome 4', 6'-diamidino-2-phenylindole (DAPI). The frequency of MN in slides stained with DAPI was significantly higher than in those stained with Giemsa, which might be due to an underestimation of small micronuclei in Giemsa-stained slides.     

Determination of restriction enzyme activity when cutting DNA labeled with the TOTO dye family

Optical mapping, a single DNA molecule genome analysis platform that can determine methylation profiles, uses fluorescently labeled DNA molecules that are elongated on the surface and digested with a restriction enzyme to produce a barcode of that molecule. Understanding how the cyanine fluorochromes affect enzyme activity can lead to other fluorochromes used in the optical mapping system. The effects of restriction digestion on fluorochrome labeled DNA (Ethidium Bromide, DAPI, H33258, EthD-1, TOTO-1) have been analyzed previously. However, TOTO-1 is a part of a family of cyanine fluorochromes (YOYO-1, TOTO-1, BOBO-1, POPO-1, YOYO-3, TOTO-3, BOBO-3, and POPO-3) and the rest of the fluorochromes have not been examined in terms of their effects on restriction digestion. In order to determine if the other dyes in the TOTO-1 family inhibit restriction enzymes in the same way as TOTO-1, lambda DNA was stained with a dye from the TOTO family and digested. The restriction enzyme activity in regards to each dye, as well as each restriction enzyme, was compared to determine the extent of digestion. YOYO-1, TOTO-1, and POPO-1 fluorochromes inhibited ScaI-HF, PmlI, and EcoRI restriction enzymes. Additionally, the mobility of labeled DNA fragments in an agarose gel changed depending on which dye was intercalated.     

Intercalating fluorescence dye YOYO-1 prevents the folding transition in giant duplex DNA

Recently, it has become clear that with the addition of polyamines, giant DNA molecules of size greater than 10 kbp exhibit all-or-none switching between elongated coil and folded compact states. Here the effects of the intercalating fluorescent labeling dye, YOYO-1, and the minor-groove binding fluorescent labeling dye, DAPI, on the folding transition of single giant T4 DNA (166 kbp) induced by spermidine(3+) were examined, by use of the experimental technique of single molecular chain observation with fluorescence microscopy. It is found that the intercalating dye, YOYO-1, markedly prevents the folding transition, whereas the minor-groove binding dye, DAPI, exhibits negligible effect on the folding transition. This action of YOYO-1 is discussed in relation to the biological effect of intercalators.