Photosensibilisation des constituants des acides nucleiques par la proflavine. Mecanisme de formation de radicaux d''addition d''hydrogene en solutions aqueuses gelees

Photosensitization of constituents of nucleic acids by proflavine. Mechanism of formation of hydrogen addition radicals in frozen aqueous solutions

It is shown that the insertion of nucleotides between proflavine molecules is favourable to photosensitization. Furthermore, (1) each molecule of proflavine gives at the most one free radical in the substrate, (2) the chromophore is largely restored when oxygen is not present, (3) superoxide radicals are observed in the presence of oxygen, and (4) formyl radicals are detected. The scheme elaborated for the mechanism gives an explanation for all these observations.     

Stabilization of the T-state of human hemoglobin by proflavine, an antiseptic drug.

The effect of proflavine (3,6-diaminoacridine), an antiseptic drug, on the spectroscopic and oxygen binding properties of ferrous human adult hemoglobin (Hb) has been investigated. Upon binding of proflavine to the nitric oxide derivative of ferrous human adult hemoglobin (HbNO), the X-band EPR spectrum displays the characteristics which have been attributed to the T-state of the ligated tetramer. In parallel, oxygen affinity for the deoxygenated derivative of ferrous human adult Hb decreases in the presence of proflavine. The effect of proflavine on the spectroscopic and ligand binding properties of ferrous human adult Hb is reminiscent that of 2,3-D-glycerate bisphosphate, the physiological modulator of Hb action.     

Photodynamic activity of acridine orange: peroxide radical induction in DNA and synthetic polynucleotides

As shown by electron paramagnetic resonance, acridine orange induces the formation of peroxide radicals in DNA when dye-DNA mixtures frozen at 77 K are irradiated with visible light. The reaction is oxygen dependent and strongly reduced by the addition of an electron scavenger. Factors of the medium can modulate the reaction: an ionic strength increased up to 0.3 greatly enhances the dye efficiency whereas the presence of phosphate ions has an inhibiting influence. Acridine orange, which is slightly less efficient than proflavine on native DNA, induces an important peroxide radical formation in poly(dG).poly(dC) but has no action on the poly(dA).poly(dT)polymer.     

Visible-light-induced OH radicals in DNA-proflavine complexes: an e.p.r. and spin trapping study

Using the spin-trapping technique, irradiation with visible light of complexes between DNA and proflavine was shown to generate OH radicals. The characteristic spectra were not obtained when proflavine or DNA were irradiated alone, nor when oxygen was absent. Using DMPO as a spin trap we found that the intensity of the DMPO-OH e.p.r. signal was enhanced when the molar ratio between bound proflavine and the DNA phosphorus increased up to a value of 0.15 demonstrating the efficiency of the intercalated dye molecules. A strong decrease of the e.p.r. signal was observed in the presence of various OH. scavengers like t-butanol, isopropanol or sodium benzoate. The OH. production was also decreased when the irradiation was made in the presence of SOD, ceruloplasmin or catalase and after addition of Chelex 100 resin.     

Superoxide dismutase protects against paraquat-mediated dioxygen toxicity and mutagenicity: studies in Salmonella typhimurium

Paraquat is univalently reduced to the relatively stable, but oxygen-sensitive, paraquat radical (PQ.+). This PQ.+ can react with dioxygen to generate the superoxide radical, which can further generate other more deleterious species of oxygen free radicals (i.e., hydroxyl radical, OH.). These oxygen free radicals are known to cause chromosomal breaks; therefore, it was logical to postulate that paraquat is a mutagen. This proved to be the case when tested in a modified Ames test using a liquid incubation assay. Salmonella typhimurium strains TA98 and TA100 were grown in the presence of various concentrations of PQ, as well as in the presence of known mutagenic compounds: mitomycin C, azide, and proflavine. Paraquat was much more toxic and mutagenic in a simple nutritionally restricted medium than in a rich complex medium and these toxic and mutagenic effects were oxygen dependent. Furthermore, cells containing high levels of superoxide dismutase were more resistant to the toxic and mutagenic effects of paraquat than were cells containing a normal level of this enzyme.     

Photodynamic mutagenic action of acridine compounds on yeast Saccharomyces cerevisiae

The photodynamically produced mutagenicity and toxicity of 8 acridine compounds were compared in Saccharomyces cerevisiae under resting and growing conditions. Without irradiation none of the acridines induced respiratory-deficient ('petite') colonies, indicative of mitochondrial DNA damage, in resting cells; and only acriflavine and proflavine induced 'petites' in growing cells. Also, without irradiation none of the acridines were significantly toxic or mutagenic for nuclear DNA under resting or growing conditions. However, with irradiation, acriflavine, proflavine, acridine yellow and rivanol became effective 'petite'-inducing mutagens and highly toxic for resting cells, while acriflavine, proflavine, and acridine orange became effective nuclear mutagens for resting cells. Acridine, quinacrine and 9-aminoacridine were not at all biologically effective with irradiation for resting cells. The results presented here indicate that singlet oxygen is generated by a photodynamic mechanism when acriflavine is irradiated, and further, that acridine, quinacrine and 9-aminoacridine are ineffective photosensitizers, because they are incapable of generating singlet oxygen with irradiation.     

Electron transfer between protein and DNA in gamma-irradiated deoxyribonucleoprotein.

When deoxyribonucleoprotein-proflavine complexes were studied by electron spin-resonance spectroscopy following gamma-irradiation, it was found that stable free radicals were not formed at random on the complex but were preferentially located on proflavine. Since proflavine intercalalated to DNA bases serves as a final acceptor of electrons liberated by ionization, the result of our experiment was regarded as suggesting that the electron transfer from the protein moiety to the DNA moiety occurred in the irradiated deoxyribonucleoprotein.     

活性氧、自由基与植物的衰老

介绍近 1 0年来有关活性氧、自由基的产生 ,对植物的伤害及植物对活性氧、自由基清除的研究进展。     

Peroxyl adduct radicals formed in the iron/oxygen reconstitution reaction of mutant ribonucleotide reductase R2 proteins from Escherichia coli

Catalytically important free radicals in enzymes are generally formed at highly specific sites, but the specificity is often lost in point mutants where crucial residues have been changed. Among the transient free radicals earlier found in the Y122F mutant of protein R2 in Escherichia coli ribonucleotide reductase after reconstitution with Fe2+ and O2, two were identified as tryptophan radicals. A third radical has an axially symmetric EPR spectrum, and is shown here using 17O exchange and simulations of EPR spectra to be a peroxyl adduct radical. Reconstitution of other mutants of protein R2 (i.e. Y122F/W48Y and Y122F/W107Y) implicates W48 as the origin of the peroxyl adduct. The results indicate that peroxyl radicals form on primary transient radicals on surface residues such as W48, which is accessible to oxygen. However, the specificity of the reaction is not absolute since the single mutant W48Y also gives rise to a peroxyl adduct radical. We used density functional calculations to investigate residue-specific effects on hyperfine coupling constants using models of tryptophan, tyrosine, glycine and cysteine. The results indicate that any peroxyl adduct radical attached to the first three amino acid alpha-carbons gives similar 17O hyperfine coupling constants. Structural arguments and experimental results favor W48 as the major site of peroxyl adducts in the mutant Y122F. Available molecular oxygen can be considered as a spin trap for surface-located protein free radicals.     

Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1

Rev is an essential regulatory HIV-1 protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome, activating the switch between viral latency and active viral replication. Previously, we have shown that selective incorporation of the fluorescent probe 2-aminopurine (2-AP) into a truncated form of the RRE sequence (RRE-IIB) allowed the binding of an arginine-rich peptide derived from Rev and aminoglycosides to be characterized directly by fluorescence methods. Using these fluorescence and nuclear magnetic resonance (NMR) methods, proflavine has been identified, through a limited screen of selected small heterocyclic compounds, as a specific and high-affinity RRE-IIB binder which inhibits the interaction of the Rev peptide with RRE-IIB. Direct and competitive 2-AP fluorescence binding assays reveal that there are at least two classes of proflavine binding sites on RRE-IIB: a high-affinity site that competes with the Rev peptide for binding to RRE-IIB (K(D) approximately 0.1 +/- 0.05 microM) and a weaker binding site(s) (K(D) approximately 1.1 +/- 0.05 microM). Titrations of RRE-IIB with proflavine, monitored using (1)H NMR, demonstrate that the high-affinity proflavine binding interaction occurs with a 2:1 (proflavine:RRE-IIB) stoichiometry, and NOEs observed in the NOESY spectrum of the 2:1 proflavine.RRE-IIB complex indicate that the two proflavine molecules bind specifically and close to each other within a single binding site. NOESY data further indicate that formation of the 2:1 proflavine.RRE-IIB complex stabilizes base pairing and stacking within the internal purine-rich bulge of RRE-IIB in a manner analogous to what has been observed in the Rev peptide.RRE-IIB complex. The observation that proflavine competes with Rev for binding to RRE-IIB by binding as a dimer to a single high-affinity site opens the possibility for rational drug design based on linking and modifying it and related compounds.     

Terminations of DNA synthesis on 'proflavine and light'-treated phi X174 single-stranded DNA

Bacteriophage phi X174 single-stranded DNA molecules were primed with five different restriction fragments and irradiated with visible light in the presence of proflavine. This photodamaged DNA was used as template for the in vitro complementary chain synthesis by E. coli DNA polymerase I (Klenow fragment). Chain terminations were observed by polyacrylamide gel electrophoresis of the synthesized products and localized by comparison with standard sequencing performed simultaneously on the untreated template. 90% of the chain terminations occurred one nucleotide before a guanine residue in the template strand. More than 80% of the sequenced guanine residues were blocking lesions demonstrating the absence of 'hot-spots' for the photodamaging effect of proflavine. At a defined position, the chain termination frequency increased linearly with the irradiation time and was directly influenced by the proflavine concentration present. An important part of lesions resulted from the action of singlet oxygen produced by excited proflavine as shown by the effect that both NaN3 and 2H2O exerted on the reaction. The induced blocking lesions must be important in vivo since no complete replicative forms could be extracted from cell infected with bacteriophages inactivated by 'proflavine and light' treatment.     

New sensitive agents for detecting singlet oxygen by electron spin resonance spectroscopy.

Free radicals are well-established transient intermediates in chemical and biological processes. Singlet oxygen, though not a free radical, is also a fairly common reactive chemical species. It is rare that singlet oxygen is studied with the electron spin resonance (ESR) technique in biological systems, because there are few suitable detecting agents. We have recently researched some semiquinone radicals. Specifically, our focus has been on bipyrazole derivatives, which slowly convert to semiquinone radicals in DMSO solution in the presence of potassium tert-butoxide and oxygen. These bipyrazole derivatives are dimers of 3-methyl-1-phenyl-2-pyrazolin-5-one and have anti-ischemic activities and free radical scavenging properties. In this work, we synthesized a new bipyrazole derivative, 4,4'-bis(1p-carboxyphenyl-3-methyl-5-hydroxyl)-pyrazole, DRD156. The resulting semiquinone radical, formed by reaction with singlet oxygen, was characterized by ESR spectroscopy. DRD156 gave no ESR signals from hydroxyl radical, superoxide, and hydrogen peroxide. DRD156, though, gives an ESR response with hypochlorite. This agent, nevertheless, has a much higher ability to detect singlet oxygen than traditional agents with the ESR technique.     

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.     

Extrinsic Cotton effects of proflavine bound to polynucleotides

The magnitude of the Cotton effect of proflavine which is bound to RNA or to denatured DNA depends on the ratio of bound proflavine to nucleic acid base. A statistical treatment which explains this behavior has been fitted to the experimental curves and indicates that optical activity arises through interaction between two or more bound proflavine molecules. The corresponding requirement with double helical DNA is for interaction between 3–4 proflavine molecules. Although proflavine binds to denatured DNA at pH 2.8, as shown by the shift of the proflavine spectrum, the strong binding process is absent, and to this is attributed the absence of the Cotton effect at low pH. Studies on the Cotton effects of proflavine bound to poly A and poly U at neutral pH, to poly A at acid pH and to poly (A + U) allow the generalization that a relatively rigid configuration of the binding macromolecule is required for the induction of these extrinsic Cotton effects.     

Chemical and biochemical aspects of superoxide radicals and related species of activated oxygen

The spectrum of biological processes in which oxygen is used by living systems is quite large, and the products include some damaging species of activated oxygen, particularly the superoxide radical (O-.2) and hydrogen peroxide (H2O2). Superoxide radicals and hydrogen peroxide, in turn, can lead to the formation of other damaging species: hydroxyl radicals (.OH) and singlet oxygen (1O2). Hydroxyl radicals react with organic compounds to give secondary free radicals that, in the presence of oxygen, yield peroxy radicals, peroxides, and hydroperoxides. Formation, interconversion, and reactivity of O-.2 and related activated oxygen species, methods available for their detection, and the basis of their biological toxicity are briefly reviewed.     

Comparative studies of the effects of acridines and other petite inducing drugs on the mitochondrial genome of Saccharomyces cerevisiae.

Summary The effects of the acridines euflavine and proflavine on mitochondrial DNA (mtDNA) replication and mutation inSaccharomyces cerevisiae have been compared. In contrast to previous results we found that under our conditions proflavine can indeed induce high levels (>80%) of petite mutants, although six times less efficiently than euflavine. The parameters measured for mutagenesis of the mitochondrial genome and inhibition of mtDNA replication in whole cells suggest that the modes of action of euflavine and proflavine are very similar. After extended (18h) treatment of growing cells with each drug the percentage loss of mtDNA or genetic loci was almost coincidental with the extent of petite induction.It was found that proflavine is equally as effective as euflavine in inhibiting mtDNA replication in isolated mitochondria in contrast to the differential between the drugs observed in vivo. However, proflavine and euflavine inhibit cellular growth at almost the same concentrations. It is therefore proposed that there is some intracellular permeability barrier which impedes proflavine access to the mitochondrial DNA replicating system.The petites induced by euflavine (and proflavine) are characterized by there being a preferential induction ofrho ⁰ petites lacking mtDNA as opposed torho ^- petites retaining mtDNA. This is in contrast to the relative proportions of such petites induced by ethidium bromide or berenil. A scheme for the production of petites by euflavine is presented, in which euflavine inhibits the replication of mtDNA, but does not cause direct fragmentation of mtDNA (unlike ethidium bromide and berenil). The proposed scheme explains the production of the high frequency ofrho ^o cells, as well as therho ^- cells induced by euflavine. The scheme also accounts for previous observations that euflavine only mutants growing cultures, and that the buds, but not mother cells, become petite.     

Induction of cytoplasmically inherited respiration-deficient ('petite') mutants by photodynamic action of acridine compounds

All acridines used (acriflavine, proflavine, acridine orange and 3-azido-10-methylacridinium chloride) produced killing in yeast cells when activated with visible light. Acriflavine, proflavine and 3-azido-10-methylacridinium chloride, but not acridine orange, produced petite and sectored colonies. Both cell killing and petite induction by light activation of acriflavine resulted apparently from photodynamic action mediated by singlet oxygen (1O2) since the effect were prevented by either sodium azide or anaerobiosis. The biological effects of 3-azido-10-methylacridinium chloride, which was developed as a potential photoaffinity probe for studying the binding and biological effects of acridines, appeared to be due to a photodynamic action analogous to that of acriflavine. Sodium azide or anaerobiosis prevented the light-activated effects of 3-azido-10-methylacridinium chloride despite the fact that the initial chemical breakdown of the azido derivative induced by light was not affected. Cells suspended in D2O demonstrated an enhanced response to 3-azido-10-methylacridinium chloride with irradiation. These results indicate that singlet oxygen mediates the light-activated biological effects of both acriflavine and 3-azido-10-methylacridinium chloride.     

Proflavine-mediated inactivation of Salmonella dublin exposed to visible sunlight in natural fresh water

The survival of Salmonella dublin exposed to visible sunlight, and heterotrophic bacteria in freshwater microcosms in the presence and absence of the photosensitizer proflavine, was studied. Enumeration of S. dublin and the heterotrophic bacteria showed that in both illuminated and nonilluminated systems (without proflavine) the bacteria remained viable and culturable for at least 6 days. The optimal proflavine concentration (no effect in the dark and a maximal photoinactivation of salmonellae after irradiation) was 2 mg l(-1). In contrast to S. dublin, the heterotrophic bacteria overcame the initial inhibitory effect of proflavine. The possible use of photosterilization against contamination with pathogenic bacteria in water model ecosystems, is discussed.     

Effect of normothermic ischemic cardiac arrest and of reperfusion on the free oxygen radical scavenger enzymes and xanthine oxidase (a generator of superoxide anions)

Recent evidence suggests that free oxygen radicals are produced by ischaemic tissues, accounting for at least part of the damage that results. These free oxygen radicals are produced by xanthine oxidase, amongst others, and removed by scavenger enzymes (catalase, superoxide dismutase and glutathione peroxidase) and anti-oxidants. As mitochondria are oxygen-utilising organelles, they are capable of producing free oxygen radicals. Our results indicate that the removal of free oxygen radicals are not diminished during ischaemia, but the activity of the free oxygen radical generator, xanthine oxidase, is increased. This could lead to an increased superoxide anion concentration.