Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA

The fluorescent intercalation complex of ethidium bromide with DNA was used as a probe to demonstrate damage in the base-pair region of DNA, due to the action of superoxide radicals. The O.2- radical itself, generated by gamma-radiolysis of oxygenated aqueous Na-formate solutions, is rather ineffective with respect to impairment of DNA. Copper(II) ions, known to interact with DNA by coordinate binding at purines, enhance the damaging effect of O.2-. Addition of H2O2 to the DNA/Cu(II) system gives rise to further enhancement, so that DNA impairment by O.2- becomes comparable to that initiated by .OH radicals. These results suggest that the modified, Cu(II)-catalysed, Haber-Weiss process transforms O.2- into .OH radicals directly at the target molecule, DNA-Cu2+ + O.2-----DNA-Cu+ + O2 DNA-Cu+ + H2O2----DNA...OH + Cu2+ + OH- in a "site-specific" mechanism as proposed for other systems (Samuni et al. 1981; Aronovitch et al. 1984). Slow DNA decomposition also occurs without gamma-irradiation by autocatalysis of DNA/Cu(II)/H2O2 systems. In this context we observed that Cu(II) in the DNA-Cu2+ complex (unlike free Cu2+) is capable of oxidizing Fe(II) to Fe(III), thus the redox potential of the Cu2+/Cu+ couple appears to be higher than that of the Fe3+/Fe2+ couple when the ions are complexed with DNA. Metal-catalysed DNA damage by O.2- also occurs with Fe(III), but not with Ag(I) or Cd(II) ions. It was also observed that Cu(II) ions (but neither Ag(I) nor Cd(II] efficiently quench the fluorescence of the intercalation complex of ethidium bromide with DNA.     

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.     

Preparative separation of DNA-ethidium bromide complexes by zonal density gradient centrifugation

Ethidium bromide-DNA complexes separated by rate-zonal sedimentation through a density gradient can be readily visualized and purified with little cross-contamination. The method is simple, rapid, and efficient.     

Monte carlo simulations of site-specific radical attack to DNA bases

An atomistic biophysical model permitting the calculation of initial attacks to a 38-bp representation of B-DNA base moieties by water radicals is presented. This model is based on a previous radiation damage model developed by Aydogan et al. (Radiat. Res. 157, 38-44, 2002). Absolute efficiencies for radical attack to the 38-bp DNA molecule are calculated to be 41, 0.8 and 15% for hydroxyl radical ((.)OH), hydrogen radical (H(.)), and hydrated electron (e(aq))(,) respectively. Among the nucleobases, guanine is found to have the highest percentage (.)OH attack probability at 36%. Adenine, cytosine and thymine moieties have initial attack probabilities of 24, 18 and 22%, respectively. A systematic study is performed to investigate (.)OH attack probabilities at each specified attack site in four molecular models including free bases, single nucleotides, single base pairs, and the central eight base pairs of the 38-bp DNA molecule. Cytosine is the free base moiety for which the closest agreement is observed between the model prediction and the experimental data. The initial (.)OH attack probabilities for cytosine as the free base are calculated to be 72 and 28%, while experimental data are reported at 87 and 13% for the C5 and C6 positions on the base, respectively. In this study, we incorporated atomic charges to scale the site-specific (.)OH reaction rates at the individual atomic positions on the pyrimidine and purine bases. Future updates to the RIDNA model will include the use of electron densities to scale the reaction rates. With respect to reactions of the aqueous electron with DNA, a comparison of the initial distribution of electron attack sites calculated in this study and experimental results suggests an extremely rapid and extensive redistribution of the e(-)(aq) after their initial reactions with DNA.     

Pulse fluorimetry study in polarized light of DNA-ethidium bromide complexes

In previous works, a quantitative analysis of the fluorescence anisotropy decay, based on a comparison of the experimental measurements with a Monte Carlo simulation of the excitation energy migration, has been shown to provide the value of the unwinding angle of the DNA helix, induced by an ethidium bromide (E.B.) molecule intercalation. In the present work some of the characteristics of the model used in the computation are reexamined: namely the influence of the direction of the E.B. electronic moment, and the influence of the dye distribution along the DNA helix are studied. The computations are compared with experimental results obtained with new experiments performed with calf thymus and micrococcus lysodeikticus DNA-E.B. complexes. It is found that the difference in base composition of these DNA does not influence the fluorescence properties of their E.B. complexes. Our study confirms the validity of the dye distribution obtained with the single adjacent excluded site principle. Reasonable values of the unwinding angle are obtained by assuming that the transition moment direction lies along the great axis of the E.B. molecule. The value of this unwinding angle is compared with other values proposed in the literature.     

Influence of the DNA structure on the free radical induction due to proflavine and light treatment

Summary Induction of peroxide free radicals (detected by Electron Paramagnetic Resonance at 77 K) due to the photodynamic activity of proflavine was measured on bacteriophage X174 DNA either single-stranded (ss) as isolated from the virion, or double-stranded supercoiled (RFI) as isolated from the infected bacteria. Comparison was made with calf thymus DNA photosensitization.In order to use equivalent DNA-proflavine complexes, binding of the dye to the three DNA's was first determined under those conditions of high ionic strength favourable to the photodynamic reaction.Free radical induction was maximal for definite amounts of bound proflavine (which varied depending upon the DNA substrate) and at an ionic strength value of 0.5. The level of the maximal reaction increased in the following order: from Xss DNA to calf thymus DNA and finally to XRFI DNA. The conformation of the proflavine-DNA complex was thus a determinant for the efficiency of the photodynamic process. The ionic strength effect could not be explained by the evolution of the proflavine triplet state in irradiated proflavine-calf thymus DNA complexes.     

A new biophysics approach using photoacoustic spectroscopy to study the DNA-ethidium bromide interaction

We have examined the binding processes of ethidium bromide interacting with calf thymus DNA using photoacoustic spectroscopy. These binding processes are generally investigated by a combination of absorption or fluorescence spectroscopies with hydrodynamic techniques. The employment of photoacoustic spectroscopy for the DNA-ethidium bromide system identified two binding manners for the dye. The presence of two isosbestic points (522 and 498 nm) during DNA titration was evidence of these binding modes. Analysis of the photoacoustic amplitude signal data was performed using the McGhee-von Hippel excluded site model. The binding constant obtained was 3.4 x 10(8) M(bp)(-1), and the number of base pairs excluded to another dye molecule by each bound dye molecule (n) was 2. A DNA drug dissociation process was applied using sodium dodecyl sulfate to elucidate the existence of a second and weaker binding mode. The dissociation constant determined was 0.43 mM, whose inverse value was less than the previously obtained binding constant, demonstrating the existence of the weaker binding mode. The calculated binding constant was adjusted by considering the dissociation constant and its new value was 1.2 x 10(9) M(bp)(-1) and the number of excluded sites was 2.6. Using the photoacoustic technique it is also possible to obtain results regarding the dependence of the quantum yield of the dye on its binding mode. While intercalated between two adjacent base pairs the quantum yield found was 0.87 and when associated with an external site it was 0.04. These results reinforce the presence of these two binding processes and show that photoacoustic spectroscopy is more extensive than commonly applied spectroscopies.     

RADACK, a stochastic simulation of hydroxyl radical attack to DNA

RADACK was conceived to simulate the radiation-induced attack to different DNA forms and complexes. It allows to separately calculate the probability of attack to each reactive atom of the sugar and of the base and takes into account the sequence-dependent structure of DNA as known from crystallographic or NMR studies or resulting from molecular modelling. The calculations are aimed to assess sequence-, structure- and ligand-dependent modulation of damages of sugar and bases, leading to single strand breaks (frank strand breaks, FSB) and alkali-labile base modifications (alkali-revealed breaks, ARB), respectively. The modelling procedure and the results of simulations for some representative structures (B, Z and quadruplex forms) are here described and discussed. The calculated relative probabilities of OH* radical attack to all reaction sites are compared to experimental FSB and ARB values. By a fitting procedure, the relative efficiencies of conversion of the C4' and C5'-centred radicals into FSB, epsilon (C4'): epsilon (C5'), and the relative efficiencies of base radicals- to- ARB conversion, epsilon(T) : epsilon(A) : epsilon(C) : epsilon(G), are then deduced for each DNA form. The ability of the model to account for the distribution of damages in DNA-ligand complexes is proven by its successful application to two DNA-protein systems : the lac repressor-lac operator complex and the nuclcosome core.     

F2-dihomo-isoprostanes arise from free radical attack on adrenic acid

Unlike F4-neuroprostanes (F4-NeuroPs), which are relatively selective in vivo markers of oxidative damage to neuronal membranes, there currently is no method to assess the extent of free radical damage to myelin with relative selectively. The polyunsaturated fatty acid adrenic acid (AdA) is susceptible to free radical attack and, at least in primates, is concentrated in myelin within white matter. Here, we characterized oxidation products of AdA as potential markers of free radical damage to myelin in human brain. Unesterified AdA was reacted with a free radical initiator to yield products (F2-dihomo-IsoPs) that were 28 Da larger than but otherwise closely resembled F2-isoprostanes (F2-IsoPs), which are generated by free radical attack on arachidonic acid. Phospholipids derived from human cerebral gray matter, white matter, and myelin similarly oxidized ex vivo showed that the ratio of esterified F2-dihomo-IsoPs to F4-NeuroPs was approximately 10-fold greater in myelin-derived than in gray matter-derived phospholipids. Finally, we showed that F2-dihomo-IsoPs are significantly increased in white matter samples from patients with Alzheimer's disease. We propose that F2-dihomo-IsoPs may serve as quantitative in vivo biomarkers of free radical damage to myelin from primate white matter.     

Peritoneal resorption of ethidium bromide, free or linked to DNA in rodents]

Ethidium bromide, either free (EB) or bound to DNA (EB-DNA), is injected into the peritoneal cavity of adult rats or mice. EB is then detected by fluorescence microscopy in peritoneal cells and by spectrophotometry in the peritoneal fluid. EB-DNA persists for a longer period of time in the peritoneal cavity than free EB does.     

Characterization of hydroxyl free radical mediated damage to plasmid pBR322 DNA

We have investigated hydroxyl free radical mediated damage to pBR322 DNA produced by ascorbate/iron and oxygen in a phosphate-buffered in vitro system. An observed lag phase in DNA nicking suggests a multi-target model of hydroxyl free radical attack on DNA. In the present report we further examine the model system and show that there is a "heat labile" component of the ascorbate/iron system which can be completely restored by the readdition of ascorbate. These observations have allowed us to rule out the possibility that intermediates build up in the reaction and act independently of ascorbate to increase the reaction rate. We have investigated the initial rate of OH production with two OH trapping agents, salicylate and deoxyguanosine, and find that the lag in DNA nicking is not due to a corresponding lag in the production of OH as assessed by formation of the products, dihydroxybenzoic acids and 8-hydroxydeoxyguanosine, respectively. We have found that the energy of activation for DNA supercoiled nicking is 13.9 kcal/mole and for OH trapping by salicylate is 21.1 kcal/nmole. These two activation energies are sufficiently different to suggest that the rate-limiting steps of these two reactions are different. Investigation of the rate of oxygen consumption during the ascorbate/iron-mediated DNA damage showed that oxygen was not a limiting component at any point in the reaction. The addition of catalase slowed down oxygen consumption by 31% and this data taken together with our previous observations on the model implicate hydrogen peroxide as a key intermediate in DNA damage caused by hydroxyl free radical.     

Iron-dependent free radical damage to DNA and deoxyribose. Separation of TBA-reactive intermediates

1. Iron-dependent free radical damage to DNA and deoxyribose results in the formation of thiobarbituric acid (TBA) reactive intermediates. 2. These intermediates have been compared chromatographically and spectrophotometrically after incubation with the enzymes xanthine oxidase and peroxidase. 3. Loss of TBA-reactivity occurred in the bleomycin-iron(II) derived products incubated with xanthine oxidase and in a standard solution of sodium malondialdehyde incubated with peroxidase.     

Inhibition of free radical-induced DNA damage by uric acid

Single-strand DNA breaks were produced in isolated rat liver nuclei incubated with 3 separate oxygen free radical generating systems: xanthine oxidase-acetaldehyde plus Fe(II); hematin-R(H)OOH; Fe(II)-H2O2. Uric acid inhibited the induction of damage in the first two systems only. At concentrations below those found in human plasma, it was particularly effective against strand breaks produced by hematin-cumene hydroperoxide. These results offer additional evidence that uric acid may function as a cellular protective agent against superoxide and hydroperoxyl free radical-induced cytotoxicity toxicity.     

Energetic and binding properties of DNA upon interaction with dodecyl trimethylammonium bromide.

The interaction of dodecyl trimethylammonium bromide (DTAB), a cationic surfactant, with calf thymus DNA has been studied by various methods, including potentiometric technique using DTAB-selective plastic membrane electrode at 27 and 37 degreesC, isothermal titration microcalorimetry and UV spectrophotometry at 27 degreesC using 0.05 M Tris buffer and 0.01 M NaCl at pH 7.4. The free energy is calculated from binding isotherms on the basis of Wyman binding potential theory and the enthalpy of binding according to van't Hoff relation. The enthalpy of unfolding has been determined by subtraction of the enthalpy of binding from the microcalorimetric enthalpy. The results show that, after the interaction of first DTAB molecule to DNA (base molarity) through the electrostatic interaction, the second DTAB molecule also binds to DNA through electrostatic interaction. At this stage, the predom-inant DNA conformational change occurs. Afterwards up to 20 DTAB molecules, below the critical micelle concentration of DTAB, bind through hydrophobic interactions.     

Direct observation of the reversible unwinding of a single DNA molecule caused by the intercalation of ethidium bromide

Ethidium bromide (EtBr) is the conventional intercalator for visualizing DNA. Previous studies suggested that EtBr lengthens and unwinds double-stranded DNA (dsDNA). However, no one has observed the unwinding of a single dsDNA molecule during intercalation. We developed a simple method to observe the twisting motions of a single dsDNA molecule under an optical microscope. A short dsDNA was attached to a glass surface of a flow chamber at one end and to a doublet bead as a rotation marker at the other end. After the addition and removal of EtBr, the bead revolved in opposite directions that corresponded to the unwinding and rewinding of a dsDNA, respectively. The amount of intercalating EtBr was estimated from the revolutions of the bead. EtBr occupied 57% of base pairs on a single dsDNA at 1 mM of EtBr, indicating that EtBr molecules could bind at contiguous sites to each other. The isotherm of intercalation showed that negative cooperativity existed between adjoining EtBr molecules. The association constant of EtBr and dsDNA (1.9 (±0.1) × 10⁵ M⁻¹) was consistent with that of previous results. Our system is useful to investigate the twisting of a single dsDNA interacting with various chemicals and biomolecules.     

Ascorbate/iron mediation of hydroxyl free radical damage to PBR322 plasmid DNA

Plasmid PBR322 DNA has been exposed to hydroxyl free radicals generated from an ascorbate/Fe system. Hydroxyl free radical scavengers as well as the iron chelator desferroxamine and catalase inhibit the DNA nicking which occurs, but superoxide dismutase had no effect. The DNA nicking was temperature dependent, occuring more rapidly at higher temperatures. The rate of DNA nicking was accelerated by the addition of hydrogen peroxide. There was an early lag phase in DNA nicking, even though the rate of hydroxyl free radical generation, as assessed by salicylate hydroxylation, showed no lag phase. It is considered that the early hydroxyl free radical damage to DNA may be biologically very important in mutagenic and carcinogenic processes.     

Increased hydrogen peroxide concentration in human tumor cells due to a nitroxide free radical.

Evidence is presented that the nitroxide free radical, TEMPO, at concentrations commonly used to prevent oxidative damage, increases the intracellular hydrogen peroxide concentration. To investigate the origin of this increased hydrogen peroxide concentration, we have incubated various human tumor cell lines with compounds interfering with the generation of active oxygen metabolites. Sodium azide, inhibitor of the respiratory chain, the iron-chelating agent desferrioxamine, superoxide dismutase and catalase had no effect on the hydrogen peroxide concentration. Metyrapone, inhibitor of the cytochrome P450 system, was demonstrated to decrease, but not completely prevent, the hydrogen peroxide production. N-ethylmaleimide, a sulphydryl-bond alkylating agent, was able to completely prevent the increased hydrogen peroxide production. We conclude that, by increasing the cellular hydrogen peroxide concentration, TEMPO exerts a pro-oxidant effect. This increase in hydrogen peroxide production seems to be mediated by the induction of oxidase activity in the cytochrome P450 system, but other cellular systems involved in electron transport may also play a role.     

Energetics of DNA intercalation reactions

Isothermal titration calorimetry has been used to determine the binding enthalpy and heat capacity change (DeltaC(p)()) for a series of DNA intercalators, including ethidium, propidium, daunorubicin, and adriamycin. Temperature-dependent binding enthalpies were measured directly for the ligands, from which DeltaC(p)() values of -140 to -160 cal mol(-)(1) K(-)(1) were calculated. Published van't Hoff plots were reanalyzed to obtain DeltaC(p)() values of -337 to -423 cal mol(-)(1) K(-)(1) for the binding of actinomycin D to several DNA oligonucleotide duplexes with defined sequences. Heat capacity changes for DNA intercalation were found to correlate with the alterations in solvent-accessible surface area calculated from available high-resolution structural data. Multiple linear regression was used to derive the relationship DeltaC(p)() = 0. 382(+/-0.026)DeltaA(np) - 0.121(+/-0.077)DeltaA(p) cal mol(-)(1) K(-)(1), where DeltaA(np) and DeltaA(p) are the binding-induced changes in nonpolar and polar solvent-accessible surface areas (in square angstroms), respectively. The DeltaC(p)() terms were used to estimate the hydrophobic contribution to intercalative binding free energies, yielding values that ranged from -11.2 (ethidium) to -30 kcal mol(-)(1) (actinomycin D). An attempt was made to parse the observed binding free energies of ethidium and propidium into five underlying contributions. Such analysis showed that the DNA binding behavior of these simple intercalators is driven almost equally by hydrophobic effects and van der Waals contacts within the intercalation site.