Characterization of Five Commercially Available Samples of Acridine Yellow

Commercial samples of acridine yellow, all labeled C.I. 46025, have been analyzed by thin layer chromatography, UV and visible light spectroscopy, mass spectrometry, and photodynamic efficiency in the inactivation of bacteriophage φX174. Three types of sample were clearly delineated: i) true acridine yellow (3,6-diamino-2,7-dimethylacridine) whose spectral and chromatographic properties are very close to those of proflavine (3,6-diaminoacridine); ii) a pure but different dye tentatively identified as euchrysine (3,6-diamino-2,7,9-trimethylacridine), since on the basis of mass spectral data, it contains an additional methyl group not fixed on the amino groups; and iii) a complex dye with its own special properties and whose main yellow component has a molecular weight and a mass spectrum compatible with an overall formula of C₁₆H₁₆N₂S. The three types of dye could be distinguished on the basis of simple tests. Acridine yellow is photodynamically almost as efficient as proflavine, but the two other dyes are very poor sensitizers.     

The induction of chromosomal aberrations and SCE by visible light in combination with dyes. I. The effect of hypoxia during light exposure in unsynchronized Chinese hamster ovary cells, sensitized with acridines

A comparison has been made between the ability of different acridine compounds to act as sensitizers for visible light (400-700 nm) induced chromosomal aberrations and sister-chromatid exchanges (SCE) in unsynchronized Chinese hamster ovary (CHO) cells. Cells were treated for 20 min with acridines (0.1-5.0 microgram/ml), washed free of excess dye and subsequently exposed to visible light (2 x 40 W/8 W m-2) either in air or in nitrogen for 5-15 min. The 4 acridines tested, proved to be effective sensitizers for the induction of both chromosomal aberrations and SCE by visible light. The most pronounced effect was observed when the light exposure of the fluorochrome-pretreated cells was performed in air. Hypoxic conditions during light exposure reduced the effect dramatically, especially in the case of induced chromosomal aberrations. The order of efficiency for the induction of both chromosomal aberrations and SCE was acridine orange greater than acridine yellow greater than proflavine greater than 3,6-diamino-10-methylacridine. The results are discussed in terms of S-independent versus S-dependent mechanisms for inducing chromosomal alterations and the potential involvement of oxygen-derived free radicals in this process.     

Production of extracellular plasmocoagulase in staphylococcus aureus exposed to 3,6-diaminoacridines]

A modified quantitative nephelometric method was used. It was shown that production of extracellular plasmocoagulase was depressed in the development of acridine-sensitive and acridine-resistant cultures of Staph. aureus in the fluid nutrient medium containing 3,6-diaminoacridines: acriflavine, proflavine, acridine yellow, acridine orange, acridine No. 40 and acridine No. 56; 3,6-diaminoacridines failed to lead to noncompetitive inhibition of staphylococcus plasmocoagulase. A disturbance of the regulation of the enzyme exit into the external environment can serve as the cause of depression of the extracellular plasmocoagulase production by Staph. aureus.     

Fluorescence decay studies of the DNA-3,6-diaminoacridine complexes

The interaction of several 3,6-diaminoacridines with DNAs of various base composition has been studied by steady-state and transient fluorescence measurements. The acridine dyes employed are of the following two classes: class I - proflavine, acriflavine and 10-benzyl proflavine; class II - acridine yellow, 10-methyl acridine yellow and benzoflavine. It is found that the fluorescence decay kinetics follows a single-exponential decay law for free dye and the poly[d(A-T)]-dye complex, while that of the dye bound to DNA obeys a two-exponential decay law. The long lifetime (tau 1) for each complex is almost the same as the lifetime for the poly[d(A-T)]-dye complex, and the amplitude alpha 1 decreases with increasing GC content of DNA. The fluorescence quantum yields (phi F) of dye upon binding to DNA decrease with increasing GC content; the phi F values for class I are nearly zero when bound to poly(dG) X poly(dC), but those for class II are not zero. This is in harmony with the finding that GMP almost completely quenches the fluorescence for class I, whereas a weak fluorescence arises from the GMP-dye complex for class II. The fluorescence spectra of the DNA-dye complexes gradually shift toward longer wavelengths with increasing GC content. In this connection, the fluorescence decay parameters show a dependence on the emission wavelength; alpha 1 decreases with an increase in the emission wavelength. In view of these results, it is proposed that the decay behavior of the DNA-dye complexes has its origin in the heterogeneity of the emitting sites; the long lifetime tau 1 results from the dye bound to AT-AT sites, while the short lifetime tau 2 is attributable to the dye bound in the vicinity of GC pairs. Since GC pairs almost completely quench the fluorescence for class I, partly intercalated or externally bound dye molecules may play an important role in the component tau 2.     

Study of the interaction of proflavin with deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in aqueous solution by uni- and two-dimensional 1H-spectroscopy]

Complex formation between acridine dye proflavine and self-complementary deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in water-salt solution was studied by the method of one- and two-dimensional 1H-NMR spectroscopy (500 MHz). Two-dimensional homonuclear 1H-NMR spectroscopy (2D-COSY and 2D-NOESY) was used for complete assignments of proton signals of molecules in solution and for qualitative analysis of the nature of interactions between proflavine and tetranucleotide. Concentration dependences of proton chemical shifts of the molecules were measured at 293 K. Equilibrium reaction constants and limiting chemical shifts of dye protons in the complexes were determined using suggested schemes of complex formation. Based on the obtained data possible types of complexes were considered. Analysis of relative content of different types of complexes was made and special features of dynamic equilibrium were revealed as a function of correlation of dye and tetranucleotide concentrations. The most favourable structure of 1:2 complex of dye with tetranucleotide was constructed using the calculated values of induced chemical shifts of proflavine protons and 2D-NOESY spectra.     

Fluorescence decay and quantum yield characteristics of acridine orange and proflavine bound to DNA

Fluorescence properties (quantum yield, decay curve, lifetime and polarization) of acridine orange and proflavine bound to DNA were examined as a function of nucleotide to dye (P/D) ratio. First, mean fluoiescence lifetimes were determined by the phase-shift measurements. The lifetime and quantum yield of acridine orange increased in a parallel fashion with increasing P/D ratio. There was no parallel relation between the lifetime and quantum yield for proflavine; the lifetime showed a minimum around P/D = 10. Next, fluorescence decay curves were measured by the monophoton counting technique and analyzed with the aid of the method of moments and the Laplace transform method. The results showed that the fluorescence decay of bound acridine orange was exponential above P/D = 10. On the other hand, the decay of bound proflavine was exponential above P/D = 100, but markedly deviated from exponentiality with decreasing P/D ratio. The results of fluorescence polarization suggested that this phenomenon is the result of Förster energy transfer between proflavine molecules bound to the fluorescent site (AT pair) and bound to the quenching site (GC pair). Critical transfer distances were 26-4 and 37.0 Å, respectively, for bound proflavine and acridine orange.     

Photocleavage of DNA by the p-nitrobenzoyl group covalently linked to proflavine.

We have synthesized two novel DNA photocleaving agents,3,6-diamino-10-[6-(4-nitrobenzoyloxy)hexyl]acridinium chloride and 3,6-diamino-10-[6-(4-nitrobenzamido)-hexyl]acridinium chloride, and studied their DNA binding mode and cleavage properties. These compounds contain the photoactive p-nitrobenzoyl group attached to proflavine via an amide or ester linker group and a polymethylene chain. Spectroscopic and viscometric studies have shown that the compounds bind DNA by an intercalative mode. The presence of covalently-bonded intercalator is essential for the UV (310 nm) induced DNA scission. Above a critical ratio, an increase in the relative concentration of compound to DNA did not induce further cleavage. The cleavage efficiency was dependent on the type of linker group. These results are discussed in regard to possible mechanisms for photoinduced DNA breakage.     

The inhibition of ribonucleic acid polymerase by acridines

1. The aminoacridines, proflavine (3,6-diaminoacridine) and 9-aminoacridine, and a hydrogenated derivative, 9-amino-1,2,3,4-tetrahydroacridine, were shown to inhibit in vitro the DNA-primed RNA polymerase of Escherichia coli. The inhibition is strong with both proflavine and 9-aminoacridine, but weak with 9-amino-1,2,3,4-tetrahydroacridine. 2. The extent to which the three acridines bind to calf-thymus DNA in the enzyme medium was studied spectrophotometrically. The extent of binding decreases in the order: proflavine, 9-aminoacridine, 9-amino-1,2,3,4-tetrahydroacridine. Some evidence was also obtained for interaction between the nucleoside triphosphate substrates and proflavine or 9-aminoacridine; no such interaction was detectable with 9-amino-1,2,3,4-tetrahydroacridine. 3. Although the amount of acridine bound to DNA increases with increasing inhibition, a stage is reached where an increase in acridine concentration still causes an increase in inhibition, with practically no increase in the amount bound to DNA. 4. Plots of reciprocal rates against the reciprocal of DNA concentration were linear and had a common intercept when proflavine or 9-aminoacridine was present. Similar relations were obtained when the reciprocal concentration of nucleoside triphosphates was plotted. The observations are interpreted kinetically in terms of a competitive inhibition of the enzyme by proflavine or 9-aminoacridine and of a kinetic role for the DNA analogous to ;activation'. 5. This suggests that inhibitory acridine molecules can occupy the sites on the RNA polymerase that are specific for binding the nucleoside triphosphate substrate or the bases of the DNA, when these become accessible during the copying process.     

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.     

DNA binding of a proflavine derivative bearing a platinum hanging residue

New platinum(II) complex of 3,6-diamine-9-[6,6-bis(2-aminohethyl)-1,6-diaminohexyl]acridine, AzaPt, has been synthesised and characterised. Behaviour of AzaPt in solution (protonation and possible self-aggregation phenomena) has been investigated by spectral methods (absorbance and fluorescence) at I = 0.1 M and 25 °C, and the equilibrium parameters of binding to calf thymus DNA have been established. Two different modes of DNA binding by the complex were detected, which depend on the polymer to dye molar ratio (P/D). At relatively low P/D values the mode was interpreted as binding by the polyamine residue external to the base pairs, while at high P/D values the binding corresponds to intercalation of the proflavine residue. Such interpretation is supported by the observed salt effect on binding and the temperature variation of the binding constants, which allowed estimating the ΔH and ΔS values contributions. Spectrophotometric analysis of the long time range binding revealed that AzaPt is involved in a slow reaction, interpreted as an attack by the platinum ion on the nucleobases. The time constant for such interaction was calculated and found to be the same order of magnitude as for processes responsible for the action of anti-tumour drugs that do covalently bind to polynucleotides.     

Factors influencing transduction of genetic determinants of penicillinase activity and pathogenicity in staphylococcus aureua. II. Antiphage activity of acridine derivatives]

Acridine dyes examined earlier (acrichine, acridine orange, proflavine and rivanol) and newly-synthesized preparations (acridines No. No. 37--40) were studied in the capacity of nonspecific agents influencing the lytic cycle in development of staphylococcus phages. Acrichine and acridine No. 37 failed to prevent lysis of the indicator staphylococcus cultures (strains 16/160 and 8325) by bacteriophages; proflavine, rivanol, acridines No. No. 39--40 produced a marked inhibitory effect; acridine orange and acridine No. 38 inhibited the staphylococcus lysis completely. Some preparations could be used to investigate the transduction phenomenon.     

The acridine dyes: Their purification,physicochemical, and cytochemical properties

Summary The present investigation was designed to allow a critical comparison of the cytochemical behaviour of commercially available acriflavine dye samples and pure acriflavine and proflavine dyes, regarding their application in automated cell analysis. Thin layer chromatography, NMR-spectroscopy and mass-spectrometry were applied for the identification of the dye composition.This study includes (1) a column chromatographic technique for the purification of larger dye quantities, (2) the investigation of the photodecomposition of different dye samples, and (3) the evaluation of the influence of various acriflavine/proflavine dye concentrations (1.6·10^–3–4·10^–6 mol/l) on to the emission spectrum of stained unhydrolyzed and hydrolyzed chicken erythrocytes.The commercially available acriflavine dye samples showed a much higher reduction in fluorescence intensity than the pure dyes, whereby proflavine faded less than acriflavine. Photodecomposition is markably influenced by dye impurities. Fluorescence emission spectra were registered at various acriflavine and proflavine dye concentrations for unhydrolyzed and hydrolyzed chicken erythrocytes in order to investigate the dye-dye interaction and the behaviour of the cellular DNA-dye complex. Proflavine showed a similar spectral behaviour as acriflavine. The dye concentration-dependent spectral behaviour of the DNA-dye complex of these fluorochromes seems to be a very critical factor. A comparison of quantitative fluorescence measurements can only be performed by staining cells with the same dye quality, because automated cytology requires reproducible information of cells in machinesensible terms.This investigation was supported by a grant from the Bundesministerium für Forschung und Technologie (01 VH 065)     

Apparent changes in structure-activity relationships for antimitochondrial effects of 9-anilinoacridines according to Saccharomyces cerevisiae strain and methodology

Sensitivity of detection of antimitochondrial effects in S. cerevisiae as measured by the induction of 'petite' mutants, has been investigated in a closely related series of 9-anilinoacridines, using a new microtitre test which has been compared to a range of other techniques. Drugs were chosen to span antimitochondrial activity between the inactive compounds 9-amino- or 3-amino-acridine and the moderately active proflavine, also between proflavine and the strong antimitochondrial agent, ethidium bromide. As previously reported using other techniques, no compound without an amino substituent caused antimitochondrial effects, whereas all 9 anilinoacridines with a 1'-substituted anilino group and 3,6-diamino-substituted acridine ring acted like ethidium in causing strong 'petite' mutagenesis. Compounds with a single acridine 3-amino group, together with proflavine, might or might not be scored as an antimitochondrial agent depending on the time and conditions of drug exposure and, more importantly, on the selection of yeast strain used in the screening. Measurement of 'petite' mutagenesis in strain 5178B, using the microtitre assay, provided the most sensitive and efficient means of detection of antimitochondrial effects for all physical DNA-binding agents. Detailed interpretation of structure-activity relationships and prediction of carcinogenic activity based upon induction of 'petite' mutagenesis would vary considerably if this procedure is not followed.     

NMR study of complex formation of aromatic ligands with heptadeoxynucleotide 5'-d(GCGAAGC) forming stable hairpin structure in aqueous solution

Complex formation of hairpin-producing heptadeoxynucleotide 5'-d(GCGAAGC) with aromatic molecules: acridine dye proflavine and anthracycline antibiotic daunomycin was studied by one-dimensional 1H NMR and two-dimensional correlation 1H-1H (2M-TOCSY, 2M-NOESY), 1H-31P (2M-HMBC) NMR spectroscopy (500 and 600 MHz) in aqueous solution. Concentration and temperature dependences for the chemical shifts of ligand protons were measured, molecular models of equilibrium in solution were developed, and equilibrium thermodynamic parameters for the formation of intercalation complexes were calculated. Spatial structures of dye and antibiotic complexes with the heptamer hairpin were constructed on the basis of 2M-NOE data and the calculated values of limiting chemical shifts of ligand protons.     

Extrinsic cotton effects of aminoacridines bound to DNA

The optical rotatory dispersion curves of the proflavine cation were measured in the spectral range 400–500 mμ. No optical activity was observed for the free cation but a large positive Cotton effect appeared in the presence of DNA. The effect of ionic strength, denaturation of the DNA, and the DNA/proflavine ratio were studied. The dependence of the magnitude of the Cotton effect on the DNA/proflavine ratio suggests that a nearest-neighbor interaction between bound proflavine molecules is necessary for optical activity. A simple statistical treatment was made which indicated that only a small number of proflavine molecules are required in close proximity for optical activity to occur. Denaturation of the DNA did not destroy the optical activity, which shows that long runs of DNA double helix are not necessary for optical activity of the ligand molecules. The optical rotatory dispersion curves of acridine orange which was bound to DNA were also measured. Two Cotton effects of opposite sense could be distinguished, the relative magnitudes of which depended on the DNA/acridine orange ratio and the state of denaturation of the DNA. The apparent differences from the proflavine-DNA system can to a large extent be explained in terms of the tendency of acridine orange to form aggregates.     

Acridine Binding by Escherichia coli: pH Dependency and Strain Differences

Acridine dye binding by cells of Escherichia coli has been characterized in terms of a number of parameters. There is a temperature-dependent, readily reversible binding of acriflavine which occurs to a greater extent with acridine-sensitive mutants of E. coli K-12 than with wild-type E. coli B or K-12. There is an essentially irreversible internal binding of acriflavine which occurs when the cellular permeability barriers are destroyed or altered by heat-treatment, elevated pH, treatment with toluene or phenethyl alcohol, or infection with bacteriophage T2 or T4. Both the reversible and the irreversible binding of acridines occurs more effectively with the acridine dye acriflavine than with the related dye proflavine, and still less effectively with 9-aminoacridine and quinacrine. These properties of acridine binding can be correlated with various inhibitory effects of the dyes on the cells.     

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.     

Mechanism of enhancement of polynucleotide binding to cells by mutagens.

The binding of polyuridylate to cells is substantially increased by proflavine. This enhanced binding is saturable with respect to time and to the concentration of both proflavine and polyuridylate. Enhancement is observed only when cells are exposed to both proflavine and polyuridylate together and depends cooperatively on the proflavine concentration. The resulting complex formed between the cell, proflavine, and polyuridylate can be dissociated with salt but not with sucrose solutions. An increase in the binding of polyuridylate to cells similar to that observed with proflavine was also obtained with cationic dyes such as acridine orange, 9-aminoacridine, and Hoechst 33258, while the introduction of a bulky polysaccharide residue, dextran, into the dyes cancels these effects. Similarly, cationic aromatic compounds such as primaquine and quinacrine which carry bulky nonplanar substituents or aliphatic cationic compounds like ethylenediamine do not enhance binding. Proflavine is unable to augment the binding of a basic macromolecule, diethylaminoethylaminoethyldextran, to cells. The model proposed for the enhanced binding of polyuridylate is based on the cooperative formation of stacked complexes of cationic dye located between the cell surface and the bound polyuridylate.     

Absorption and Fluorescence Spectra of Euchrysine Ggnx and Acridine Orange; Effects of Heparin, NaCl and a Cation Exchange Resin

The absorption and fluorescence spectra of two samples of dye labeled euchrysine were found to differ. One sample, labeled GGNX, had absorption and fluorescence maxima of 435 and 515 nanometers (nm) respectively. The other sample was not further labeled, but had absorption and fluorescence maxima of 492 and 535 nm. The latter values, as well as the shape of both the fluorescence and absorption curves of the second sample were superimposable on a recrystallized sample of acridine orange labeled correctly C. I. 46905. Euchrysine has two free amino groups which are fully methylated in acridine orange, therefore a nitrous acid test can differentiate the two dyes. The sample of euchrysine labeled GGNX gave a reaction, as did acridine yellow, C. I. 46025, but acridine orange, C. I. 46005, did not. Fluorescence metachromasy of euchrysine is less efficient than that of acridine orange in two ways: the shift in the spectrum is smaller by about 40 nm, making the separation of the colors more difficult both visually and by instruments and the metachromatic fluorescence has less than half of the intensity of acridine orange as measured at the peak for each dye. Confusion between these two dyes has occurred because suppliers have used the names interchangeably. For critical studies, the dye used should be identified by its Colour Index number.     

The Inhibition of Bacteriophage DNA Injection by Dyes Bound to the DNA

A delay (~10 min) in the appearance of intracellular phage is caused by preincubating the infecting phage T4o₁ in proflavine, acridine orange, or ethidium, but not polyamines. No significant delay in attachment is observed. Apparently the presence of the dye is required inside the permeability barrier of the phage at the time of infection. The effect of proflavine is reduced in the presence of polyamines, suggesting that the active site is on DNA. The phage-host complex is sensitive to shear if the infecting phage have been incubated in proflavine or ethidium, indicating that the completion of DNA injection is delayed. Finally no partially injected complexes could be detected after shearing, which suggests that most of the delay occurs near the beginning of the injection process.