Fluorescence decay measurements for determining the relative content of ethidium bromide to DNA in situ in cell nuclei

A fluorometric method for the determination of the amount of ethidium bromide (EB) bound to DNA in situ in cell nuclei is discussed. Even when the EB content was very small, the molar ratio of DNA-phosphorus (DNA-p) to dye (P/D ratio) could be estimated by measuring the lifetime of the transient fluorescence of the EB-DNA complex as a function of the P/D ratio. To examine the relationship between the fluorescence intensity, lifetime, and P/D ratio, polyacrylamide gel film containing 4.7 mM DNA-p was used as a model DNA tissue, and its fluorescence was measured using a nanosecond microfluorometer. The fluorescence intensity showed a maximum at P/D = 6. The fluorescence lifetime increased with the P/D ratio, and this was accompanied by a proportional increase in the quantum efficiency. Thus, the lifetime value was an effective parameter for the determination of the P/D ratio in situ in tissue. When this approach was applied to tissue sections of mouse liver treated with solutions of EB at concentrations of 10 and 50 micrograms/ml, the fluorescence lifetimes on cell nuclei were 18.9 and 17.4 ns with P/D ratios of 20 and 12, respectively, as based on the model-tissue experiments. When the P/D ratio was 20, the concentration of EB in the nucleus was approximately 1.5 mM, i.e., 60 times higher than that in the staining solution.     

Fluorescence anisotropy decay of ethidium bound to chromatin

The fluorescence anisotropy decays of the chromatin ethidium complexes have been measured in solutions in which the dye was bound to the high affinity sites of the nucleosome DNA. Energy transfers between chromatin-bound ethidium molecules cause an increase of the anisotropy decay rate for much smaller values of the concentration ratio of dye to nucleotide than in the case of nacked DNA-ethidium complexes. This result implies that the high affinity sites are clustered on a short nucleosomal DNA segment. Quantitative analysis of the experimental data by computer simulations of the energy transfer process, shows that these sites are gathered on a single nucleosomal DNA segment, 28 base pairs long. Such a segment probably belongs to the nucleosome “linker”, contributing about half of it.     

Stacking interactions of ethidium bromide bound to a polyphosphate and phage DNA in situ

Ethidium bromide forms spectroscopically detectable aggregates in aqueous solution and at a high dye concentration larger than 1 × 10^?3 moles/ρ. At moderate concentration in the order of 1 × 10^?4 moles/ρ the dye interacts with inorganic polyphosphate Graham salt and with phage sd DNA in situ by formation of stacking complexes. Maximal stacking was found at a phosphate to dye ratio, P/D, of approximately 1 for Graham salt and 1.5–2 for phages. In going to a higher P/D ratio Graham salt dye complex dissociates again and free dye reappears, while phage dye binding changes from stacking (type II complex) to intercalation (type I complex). Stacking is accompanied by a decrease and intercalation by an increase of relative fluorescence intensity with respect to free dye. However, both binding types lead to hypechromism and a red shift of the dye absorption band in the visible spectral region. Thus spectral behavior of ethidium aggregates deviate clearly from that known for other dyes, i.e., acridines.     

Fluorescence anisotropy decay due to rotational brownian motion of ethidium intercalated in double strand DNA

The transient fluorescence of solutions of ethidium bromide . DNA complexes has been measured by pulse fluorimetry at different temperatures and in solvents containing various amounts of sucrose. The molar ratio of ethidium to nucleotides was low. Under these conditions the anisotropy decay was due to the Brownian motion of ethidium molecules intercalated in the double strand DNA molecules. This anisotropy decay could be described by a sum of 3 exponential terms, with correlation times 01, 02, 03 which were linear functions of the ratio of the solvent viscosity to the absolute temperature (n/T). The amplitude of the exponential term characterized by the shortest correlation time (01) has been found to depend on temperature while the ratio of the amplitude of the two other terms (characterized by 02 and 03) was independent of temperature. These results were interpreted as follows: 01 corresponds to a fast motion of the dye in its site. 02 and 03 describe a tortional motion of the ethidium bromide. DNA complex, involving several nucleotide pairs.     

Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye

The fluorescence characteristics of ethidium bromide (Eb) complexed to calf thymus DNA have been examined using fluorescence lifetime analysis for a range of DNA (effective nucleotide concentration) to Eb molar ratios. Control of both temperature and ion concentration is necessary for reproducible analyses. Eb complexed to double stranded DNA has a maximum fluorescence lifetime of 23 ns and is easily distinguishable from a fluorescence lifetime value of 1.67 ns corresponding to unbound Eb. In a solution of calf thymus DNA containing excess Eb a binding equilibrium is reached, and this corresponds to one Eb molecule for every five nucleotides. With increasing amounts of unbound Eb, the fluorescence lifetime of the DNA-Eb complex decreases with a concomitant drop in the steady state fluorescence intensity, without a change in the amount of Eb bound to DNA. It is concluded that unbound Eb, acting via a quenching mechanism, shortens the fluorescence lifetime of bound Eb and consequently decreases the overall fluorescence intensity. This means that a different approach is necessary: time-resolved fluorescence spectroscopy directly distinguishes between a decrease in fluorescence intensity due to quenching by an excess of unbound Eb from that due to a decrease in Eb binding to double-stranded DNA. These studies suggest that techniques which measure total steady state fluorescence intensity of bound Eb in order to infer relative amounts of double-stranded DNA must be interpreted with caution. For such assays to be valid it is essential that no unbound Eb be present; otherwise a variable correction factor is required to account for unbound Eb.     

Spectrofluorometric measurement of the binding of ethidium to superhelical DNA from cell nuclei.

Structures retaining many of the morphological features of nuclei may be released by lysing HeLa cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain few chromatin proteins. We have shown that the DNA of nucleoids is quasicircular and supercoiled by measure spectrofluorometrically the amount of the intercalating dye, ethidium, bound to unirradiated and gamma-irradiated nucleoids. Ethidium binds to nucleoids in the manner characteristic of the binding to superhelical DNA: at low concentrations more ethidium binds to unirradiated nucleoids than to their gamma-irradiated counterparts with broken DNA, and at higher concentrations less ethidium binds to the unirradiated nucleoids. The quasi-circles in nucleoids are 22 times less sensitive to gamma-irradiation than are circles of pure PM2 DNA: they must contain about 2.2 X 10(5) base pairs. The constraints that maintain the quasi-circularity of nucleoid DNA are very resistant to extremes of temperature and alkali; some remain under conditions in which the duplex is denatured. The constraints are destabilised by ethidium suggesting that they are stabilised by free energy of supercoiling. Proteolytic enzymes, but not ribonucleases, remove the constraints. Possible structures for the constraining mechanism are discussed.     

Renaturation of DNA in the presence of ethidium bromide

The rate of renaturation of T2 DNA has been studied as a fuction of ethidium bound per nucleotide of denatured DNA. The Binding constants and number of binding sites for ethidium have been determined by spectral titration for denatured DNA at 55, 65, and 75°C and for native DNA at 65°C in 0.4M Na⁺. The rate of renaturation of T2 DNA was found to be independentof ethidium binding up to 0.03 moles per mole of nucleotide. Above 0.03 moles, the rate drops off precipitously approaching zero at 0.08 and 0.06 moles bound ethidium per nucleotide at 65°C respectively. A study was also made of the use of bound ethidium fluorescence as a probe for monitoring DNA renaturation reactions.     

Effects of ethidium bromide,tetramethylethidium bromide and betaine B on the ultrastructure of HeLa cell mitochondria in situ

Summary Several investigators have described the ultrastructural changes that occur in the mitochondria of cells in tissue cultures after treatment with the drug ethidium bromide (E). The mitochondria swell and the cristae become greatly altered and finally disappear; in the cristae-free region of the matrix electron-dense granules can be observed. It has been assumed that intercalation of E between the base pairs of the mitochondrial DNA induces the formation of the granular inclusions. To investigate whether intercalation is really the initial step in the generation of dense granules inside the matrix, we performed a comparative incubation study of HeLa-cell mitochondria in situ using three closely related dyes (D), i.e. E, tetramethylethidium bromide (TME) and betaine B (B). They strongly differ with regard to their affinity for DNA and their ability to cross membranes. E was used as a reference dye. TME does not intercalate, but is externally bound to DNA only weakly. The neutral B is not bound at all, but can cross membranes more easily than the cation E. Moreover, in aqueous solutions at pH7.0, B is in equilibrium with its protonated cation BH. BH and E have almost equal affinities for DNA. Therefore B may quickly pass the inner mitochondrial membranes and the cristae, and should then be bound inside the matrix, thus forming a BH-DNA complex. On the assumption that intercalation is necessary for the generation of intramitochondrial electron-dense bodies, we predicted that BH/B should be more efficient than E, while TME should be relatively ineffective. In experiments using HeLa cells, these predictions were found to be inaccurate. E, TME and BH/B produced almost the same mitochondrial alterations, but at different concentrations and after different incubation periods. In contrast to our expectations TME was much more effective than E and BH/B, with the last two behaving rather similarly.Therefore, it seems unlikely that the drugs penetrate the inner mitochondrial membrane system by simple physical diffusion or that intercalation is the preliminary step for the generation of dense granules inside the matrix. Instead, we assume that hydrophobic interaction between the dye cations E, BH and TME and the cristae is the main cause of the mitochondrial changes. The favoured binding partner of the dye cations may be the divalent anion, cardiolipin: this phospholipid is an essential part of the inner membrane system but is absent in other membranes of cells. By distributing the dyes between a lipophilic phase and water, it was shown that TME is more lipophilic than E and BH; this may explain the greater effectiveness of TME. The bound dye cations disturb the organization of the cristae, which become altered and finally disappear. We assume that the electron-dense granules in the matrix are mainly composed of the dyes and former membrane materials such as phospholipids and proteins, as well as perhaps some other hydrophobic matrix materials. This would also explain why it was impossible to digest the dense granules by DNase treatment. The drugs enter the mitochondrial matrix by disordering and finally destroying the cristae.     

Reversible tenfod reduction in mitochondrial DNA content of human cells treated with ethidium bromide

Summary Cells of the human line VA₂-B in suspension culture have been treated with very low concentrations of ethidium bromide for the purpose of reducing the amount of mitochondrial DNA (mit-DNA) per cell. Cells maintained in the presence of 5 ng/ml ethidium bromide grew at a normal rate for three days; thereafter, their doubling time gradually increased to a stable value of about 60 h. In these cells, the rate of ³H thymidine incorporation into mit-DNA decreased very rapidly to 60% of the normal, and remained thereafter at this level, while the amount of mit-DNA per cell stabilized around a level of 70–80% of the control. In cells long-term treated with 5 ng/ml ethidium bromide, the rate of mitochondrial protein synthesis was about 35% of the normal, and the cytochrome c oxidase activity about 50% of the control. Cells treated with 20 ng/ml of the drug underwent 3–4 cell doublings at control rates, then gradually stopped growing, and eventually died. In these cells, the rate of incorporation of ³H thymidine into mit-DNA was reduced to 50% of the control value after 10 min treatment with ethidium bromide, and became barely detectable after three cell doublings. At this time, the cells had on the average less than 10% of the control amount of mit-DNA, the rate of mitochondrial protein synthesis was reduced to 3% of the normal, and the specific activities of cytochrome c oxidase and rutamycin-sensitive ATPase were less than 20% of the control values. In spite of these marked changes, the cells exhibited only a 20–30% loss in cell viability, as estimated by cloning efficiency, after three days of exposure to the drug. Cells treated with ethidium bromide at 20 ng/ml for three days, and then transferred to drug-free medium, recovered a near-to-normal growth rate and cloning efficiency and a near-to-normal rate of synthesis and amount of mit-DNA in about five days.     

Pressure-jump study of the kinetics of ethidium bromide binding to DNA

Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association rate constant is k1 = 17 X 10(6) M-1 s-1, and the dissociation rate constant is k-1 = 10 s-1; in the case of poly[d(G-C)], k1 = 13 X 10(6) M-1 s-1, and k-1 = 30 s-1. From the analysis of the kinetic amplitudes, the molar volume change, delta V0, of the intercalation was calculated. In the case of poly[d(A-T)], delta V0 = -15 mL/mol, and for poly[d(G-C)], delta V0 = -9 mL/mol; that is, for both polymers, intercalation is favored as the pressure is increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of ethidium bromide to a DNA triple helix. Evidence for intercalation

The interaction of ethidium, a DNA intercalator, with the poly(dA).poly(dT) duplex and the poly (dA).2poly(dT) triplex has been investigated by a variety of spectrophotometric and hydrodynamic techniques. The fluorescence of ethidium is increased when either the duplex or triplex form is present. Binding constants, determined from absorbance measurements, indicate that binding to the triple helical form is substantially stronger than to the duplex, with a larger binding site size (2.8 base triplets compared to 2.4 base pairs). Furthermore, while binding to poly(dA).poly(dT) shows strong positive cooperativity, binding to the triplex is noncooperative. Thermal denaturation experiments demonstrate that ethidium stabilizes the triple helix. Binding to either form induces a weak circular dichroism band in the visible wavelength region, while in the region around 310 nm, there is a band that is strongly dependent on the degree of saturation of the duplex, and which is positive for the duplex but negative for the triplex. Both fluorescence energy transfer and quenching studies provide evidence of intercalation of ethidium in both duplex and triplex complexes. Binding of ethidium leads to an initial decrease in viscosity for both the duplex and triplex structures, followed by an increase, which is greater for the duplex. Taken together, these results strongly suggest that ethidium binds to the poly (dA).2poly(dT) triple helix via an intercalative mechanism.     

Covalent binding of ethidium azide analogs to Salmonella DNA in vivo: competition by ethidium bromide.

The photoreactive analogs of ethidium bromide (ethidium mono- and diazide) have been developed as drug probes to determine the actual molecular details of ethidium bromide interactions with DNA. In an effort to demonstrate that the analogs in fact mimic the parent ethidium, competition experiments were designed using ³H thymidine-labeled DNA in intact $\text{Salmonella}$ TA1538, which is reverted by the azide analogs. ¹⁴C-labeled ethidium azide analogs were used in combination with the non-labeled ethidium bromide. The results presented here demonstrate that the parent ethidium competes with the azide analogs as a DNA intercalating drug using CsCl density gradient ultracentrifugation.     

The circular dichroism in the ultraviolet of aminoacridines and ethidium bromide bound to DNA

The circular dicrosim (CD) spectra of complexes of DNA with ethidiun bromnide, profiavine, 9-aminoacridine and 4-etliyl-9-amino-acridine have been determined between 220 and 450 nm, the range lieing extended to 600 nm for ethidiufm bromide. The variation of the magnitude of the visible and near—ultraviolet CD spectra of ethidium bromide—DNA complexes with the amount of ligand bound (r) suggests a common binding position with profiavine. On the other hand, 4-ethyl-9-aminoacndine complexed to DNA shows CD spectra not distinguishable from those of 9-aminnoacnidmc in both the visible and ultraviolet. The interpretation of these results with respect to the stereochemistry of the DNA-ligand complexes is discussed.     

The DNA content in the cell nuclei of trophoblastic tumors

The DNA content in cytotrophoblast (CTB) and syncytiotrophoblast (STB) cell nuclei was assayed in tissue sections of 7 hydatidiform moles (HM) and 27 choriocarcinomas (CH). The procedure involved Feulgen's reaction and scanning cytophotometry. The analysis of summarized histograms showed the DNA distribution in CTB cell nuclei, on the one hand, and that in STB, on the other, to differ significantly in both the tumors. The HM studied cases were referred to as two subtypes on the basis of such parameters as modal class value, its ploidy and degree of nuclear poly- and heteroploidy of CTB and STB. These characteristics were used to identify three patterns of CH. A pronounced modal class (2c--4c) was typical of type 1. A wider range of modal class (2c--10c or 4c--8c) was observed in type 2. Type 3 of tumor was characterized by a pronounced polyploidy with the absence of the modal class. The analysis of individual CTB and histograms showed no significant differences between HM and CH with respect to the DNA content. An increase in the share of highly polyploid cells was associated with a shorter survival of patients.