Toxicity of Xanthene Dyes to Entomopathogenic Fungi

Effects of xanthene dyes on mycelial growth and conidial germination in three species of entomopathogenic fungi, Beauveria bassiana, Metarhizium anispoliae and Paecilomyces fumosoroseus, were evaluated in a variety of assay systems. In a disk-diffusion assay, erythrosin B and phloxine B (but not eosin B) produced zones of inhibition in colonies of all three species under continuous exposure to light at disk-loadings of 100mug. None of the dyes produced zones of inhibition in the absence of light at disk loadings of 100mug. Both erythrosin B and phloxine B inhibited mycelial growth of all three species in the light in a dose-dependent manner. Weaker dose-responses for inhibition of growth in the dark were observed for some fungus/dye combinations. Erythrosin B, tested singly, completely inhibited conidial germination in the light in all eight fungal strains tested at 100mug ml-1 medium, but failed to inhibit conidial germination in any of these strains in the dark at the same concentration of dye. For single strains of each of the three fungi, erythrosin B and phloxine B inhibited conidial germination in a dose-dependent manner in the light with IC50s < 6.2mug dye ml-1 medium for all fungus/dye combinations. Phloxine B was a more potent inhibitor of germination than erythrosin B for all three fungal species. At fixed dosages of erythrosin B and phloxine B, inhibition of conidial germination in all three species increased with time of exposure to light. These results constitute the first quantitative demonstration of photodynamic inhibition of conidial germination in fungi by xanthene dyes.     

Compatibility of Photoactive Dyes with Insect Biocontrol Agents

Integrated pest management (IPM) programmes often look for more specific ways to control pests. Biological control agents, such as the bacterium, Bacillus thuringiensis Berliner, and the fungus, Beauveria bassiana (Balsamo) Vuillemin, can control insects with minimal disturbance to the environment because of their host specificity and short half-lives. Often these agents alone cannot prevent yield loss or are too expensive. This study looked at the in vitro combination of these agents and photoactive dyes, especially phloxine B (red dye D&C 28), a Food and Drug Administration approved dye, with the intent to provide better insect control. Photoactive dyes are being tested for the control of many pest insects. Phloxine B and related xanthene dyes, eosin y, fluorescein and rose bengal inhibited the growth of both B. thuringiensis and B. bassiana . Phloxine B was the most inhibitory and fluorescein the least inhibitory dye for both microbes. The magnitude of inhibition increased with increasing concentration of dye and light intensity. Therefore, an adverse effect on the field performance of these biological control agents in combination with xanthene dyes would be expected.     

Estimation of the membrane permeability of liposomes via use of eosin Y chemiluminescence catalysed by peroxidase encapsulated in liposomes.

The initial rate of horseradish peroxidase (HRP)-catalysed chemiluminescence (CL) reaction in an aqueous compartment of liposomes was applied to the estimation of membrane permeability of liposomes. HRP-encapsulated liposomes were prepared by an extrusion method, and a CL reagent and H(2)O(2) were added into the liposomes suspensions. Fluorescein, eosin Y and phloxin B, which are xanthene dyes with different chemical structures, were used as CL reagents. Xanthene dye and H(2)O(2) permeate into the inner phase of liposomes, resulting in initiation of the HRP-catalysed xanthene dye CL reaction with H(2)O(2). The initial rate of the CL reaction was independent of the xanthene dye used. The reproducibility of the initial rate with eosin Y was better than that with fluorescein and phloxin B. When the membrane permeability of the liposomes was changed by altering the concentration of cholesterol in them, the initial rate of the eosin Y CL reaction was dependent on the membrane permeability of the liposomes.     

Investigation of staining,polarization and fluorescence microscopic properties of myoepithelial cells

Summary During studies of early arteriosclerotic lesions fibers with the staining properties of myosins were observed in epithelial cells of various organs. To obtain a basis for further studies, staining, oolarization and fluorescence microscopic properties of classical myoepithelial cells and tonofibrils were investigated. The tannic acid-phosphomolybdic acid (TP)-Levanol Fast Cyanine 5RN stain for myosins and related proteins was applied to sections of tongue and skin. In other series various milling dyes, xanthene dyes and Thiazine Red R were substituted for Levanol Fast Cyanine 5RN.Myoepithelial cells of lingual and eccrine sweat glands showed the microscopic properties of smooth muscle cells; tonofibrils had little or no affinity for the dyes tested. The terminal bar-terminal web system of glandular epithelium and the fibrous layer in ducts of eccrine sweat glands resembled myosins and differed significantly from proteins of the epiderminkeratin group, e.g. tonofibrils. In preliminary studies the iodinated xanthene dyes Rose Bengal G, Erythrosin B and Y were found suitable for light, fluorescence and electron microscopic studies.     

Synthesis and photophysical properties of new fluorinated benzo[c]xanthene dyes as intracellular pH indicators.

Two new fluorinated benzo[c]xanthene dyes were synthesized by reaction of fluorinated 1,6-dihydroxynaphthalenes with 2,4- (and 2,5)-dicarboxy-3'-dimethylamino-2'-hydroxybenzophenone. The two critical fluorinated 1,6-dihydroxynaphthalene intermediates were prepared via a regioselective route. The fluorinated benzo[c]xanthene dyes exhibit desired lower pK(a) values (6.4 and 7.2, respectively) than their parent compound (pK(a)=7.5) while the pH-dependent dual-emission characteristics are well retained. Their cell-permeable esters have been prepared for intracellular applications.     

Mutagenicity testing of certified food colors and related azo, xanthene and triphenylmethane dyes with the Salmonella/microsome system.

Thirty-seven azo, xanthene and triphenylmethane dyes including FD and C colors currently approved for use in the U.S.A. and a number of delisted food colors, were tested in the Salmonella/microsome system. In addition to direct plate tests with five tester strains (TA1535, TA100, TA1537, TA1538, TA98), the azo dyes were also assayed after chemical reduction to their component amines. Also, a selected group of azo dyes was subjected to liquid tests (both aerobic with microsomes and anaerobic) and to plate tests involving initial 16 h anaerobic incubations to facilitate microbial reduction of the azo bond. None of the presently listed FD and C colors was mutagenic in any of the test modifications. Among formerly listed colors only Butter Yellow (p-dimethylaminoazobenzene), a recognized animal carcinogen, was mutagenic in the aerobic liquid test. Several other azo dyes were either directly mutagenic, viz. Acid Alizarin Yellow R and Alizarin Yellow GG; required microsomal activation, viz. Acid Alizarin Red B and Methyl Red; or required chemical reduction and microsomal activation, viz. Acid Alizarin Violet N and Sudan IV. Of the non-azo dyes tested only two xanthene dyes appeared to be mutagenic, viz. 9-(2-sulfophenyl)-6-hydroxy-3-isoxanthenone and its 2,4,5,7-tetrabromo derivative.     

Eosin interaction of alpha-synuclein leading to protein self-oligomerization

Among various dyes including congo red, thioflavin S, thioflavin T, eosin, rhodamine 6G, and phenol red, the eosin was the only dye that induced self-oligomerization of alpha-synuclein in the presence of a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline. To analyze chemical nature of the eosin interaction with alpha-synuclein, the phenomenon of self-oligomerization was further examined with eosin congeners such as ethyl eosin, eosin B, phloxine B, erythrosin B, and rose bengal. The followings are the conclusions we have reached. First of all, intactness of the benzoate moiety of eosin and the negative charge on the carboxylic group of the dye are important factors leading to the specific interaction with alpha-synuclein. Secondly, the localized negative charge on the xanthene moiety of eosin is another critical factor for the interaction. As far as substituting halides are concerned, bromides and iodides on the xanthene moiety of the dyes do not make any difference on the alpha-synuclein interaction because both eosin and erythrosin B have induced the common phenomenon of self-oligomerization. The binding curve between eosin and alpha-synuclein was sigmoidal as the dye concentrations were increased. A double reciprocal plot of the saturation curve showed that the maximum number of eosin binding sites on alpha-synuclein was 1.85 with a dissociation constant of 390 microM. The dye binding to the protein appeared to occur via a positive cooperativity. The eosin binding site(s) was suggested to be located predominantly on the NAC region and partly related to the acidic C-terminus of alpha-synuclein. It has been, therefore, expected that this information might be useful to develop alpha-synuclein interactive molecules, which could provide eventual preventive or possible therapeutic means against various alpha-synuclein related disorders including Parkinson's disease.     

An Evaluation of the Metachromasy of Anionic Dyes. I. Visual Observations on Tissue Sections

Thirteen dyes of the azo (benzopurpurin, Congo red, trypan blue, chromotrope 2R, orange G), indigoid (indigocarmine), triphenylmethane (acid fuchsin, aniline blue, light green, methyl blue), and xanthene (eosin B, eosin Y, erythrosin B) groups were applied under standard conditions to a variety of human, rabbit, rat, mouse and frog tissues in paraffin sections. Sections were examined for color changes which might indicate metachromatic reactions analogous to the metachromasy of cationic dyes. Disazo and xanthene dyes showed shifts in hue, with some qualification on the shifts of xanthenes. Metachromatic shifts of anionic dyes were generally of low order compared to those of cationic dyes. Nuclei, erythrocytes, inner elastic laminae of arteries, keratinous structures, and certain areas in the ground substance of connective tissue most often elicited metachromasy. It is suggested that basic proteins are responsible for the metachromatic reactions. Equally interesting areas were those staining poorly (cartilage matrix, most types of mucus), since these are sites of highly acidic substances capable of binding proteins.     

Mechanism of chlorpromazine binding by Gram-positive and Gram-negative bacteria

Chlorpromazine forms charge-transfer complexes with xanthene dyes in bacteria. These complexes permit the differentiation of Gram-positive and Gram-negative bacteria in both light and polarization microscopy. The birefringence induced by the charge-transfer complex might explain the molecular basis of bacterial staining.The charge-transfer complexes formed between chorpromazine and xanthene dyes accumulate in the bacterial cell, mainly inside the bacterial cell wall. The complexes give the cells a color, which depends on the chemical composition of the staining structure, and in particular the polysaccharides of the cell wall in bacteria.Metachromatic granules were seen inside Gram-positive bacteria after chlorpromazine and rose bengal staining. Although the nature of these granules remains unclear, this type of binding may have a role in the inhibition of biochemical processes in the bacterial cells.     

Binding of rhodamine B and kiton red S to cucurbit[7]uril: density functional investigations

The binding of the laser dyes rhodamine B (RhB) and sulforhodamine B (kiton red S or KRS) to a cucurbit[7]uril (CB[7]) host has been investigated using density functional theory. Both guests (RhB and KRS) contain two N,N-diethylamino groups on a xanthene core. The lowest-energy structure of these host-guest complexes has one of the N,N-diethylamino groups encapsulated within the host cavity, that engenders C-H···O interactions with portals, while the remaining noninteracting diethylamino group resides outside the cavity. The (1)H NMR chemical shifts derived using the gauge-independent atomic orbital method are consistent with those observed in experiments.     

Oxygen-independent photocleavage of DNA by xanthene dyes.

Plasmid DNA has been efficiently photocleaved by a series of xanthene dyes in the absence of molecular oxygen. The cleavage by fluorescein proceeds mostly via its singlet excited state.     

Inhibition and enhancement of phleomycin-induced DNA breakdown by aromatic tricyclic compounds.

Cationic aromatic tricyclic compounds including triphenylmethane dyes, phenazines, phenoxazines, acridines, phenothiazines, phenanthridinium compounds, anthracenes and xanthene dyes, which amplify cell killing in phleomycin-treated Escherichia coli B cells also modified phleomycin-induced breakdown of DNA to acid-soluble fragments. A plot of DNA breakdown as a function of concentration was bell-shaped for each of the active compounds, i.e. as the concentration increased, DNA breakdown was enhanced initially, but above a certain concentration, the proportion of DNA degraded declined, often to zero. One of the compounds, acriflavine, when tested also inhibited DNA breakdown following ultraviolet irradiation. A study, by sedimentation methods, of DNA single-strand breakage in phleomycin-treated E. coli cells, using 3 representative compounds, Crystal Violet, 3,6-diaminoacridine and Methylene Blue, revealed a consistent increase in DNA strand breaks as concentration of compound increased. In similar experiments with ethidium bromide the breakage yield/concentration curve exhibited a maximum. In general, however, it seems that the inhibition of DNA-breakdown observed at higher concentrations of these amplifying compounds is not explicable by an effect on the primary breakage event, but is due to suppression of exonucleolytic activity in the cells.     

C. I. Acid Red 52: A New Stain for Cells of Granulocytic Origin

Using the xanthene dye C.I. acid red 52 (CI. 45100) as a single agent stain applied to coverslip preparations of blood and bone marrow, primary and secondary granules in cells of neutrophilic origin stained brilliant pink. In eosinophils, granules stained dark red. In leukemic myeloblasts that also stained with Sudan black B and demonstrated myeloperoxidase and specific esterase activity, a few bright red staining granules were visualized with acid red 52- In some leukemic promyelocytes, Auer rods stained bright red. In leukemic lymphoblasts, no red granules were seen. Of a wide variety of dyes tested so far, acid red 52 is the most sensitive stain for primary and secondary granules of granulocytes in blood and bone marrow.     

C. I. acid red 52: a new stain for cells of granulocytic origin

Using the xanthene dye C.I. acid red 52 (C.I. 45100) as a single agent stain applied to coverslip preparations of blood and bone marrow, primary and secondary granules in cells of neutrophilic origin stained brilliant pink. In eosinophils, granules stained dark red. In leukemic myeloblasts that also stained with Sudan black B and demonstrated myeloperoxidase and specific esterase activity, a few bright red staining granules were visualized with acid red 52. In some leukemic promyelocytes, Auer rods stained bright red. In leukemic lymphoblasts, no red granules were seen. Of a wide variety of dyes tested so far, acid red 52 is the most sensitive stain for primary and secondary granules of granulocytes in blood and bone marrow.     

Adsorption mechanism for xanthene dyes to cellulose granules

The xanthene dyes, erythrosine, phloxine, and rose bengal, were adsorbed to charred cellulose granules. The charred cellulose granules were preliminarily steeped in ionic (NaOH, NaCl, KOH, KCl, and sodium dodecyl sulfate (SDS)), nonionic (glucose, sucrose, and ethanol), and amphipathic sucrose fatty acid ester (SFAE) solutions, and adsorption tests on the dye to the steeped and charred cellulose granules were conducted. Almost none of the dye was adsorbed when the solutions of ionic and amphipathic molecules were used, but were adsorbed in the case of steeping in the nonionic molecule solutions. Thin-layer chromatography (TLC) and the Fourier transform infra-red (FT-IR) profiles of SFAE which was adsorbed to the charred cellulose granules and extracted by ethyl ether suggested the presence of hydrophobic sites on the surface of the charred cellulose granules. We confirmed that the xanthene dyes could bind to the charred cellulose granules by ionic and hydrophobic bonds.     

Adsorption Mechanism for Xanthene Dyes to Cellulose Granules

The xanthene dyes, erythrosine, phloxine, and rose bengal, were adsorbed to charred cellulose granules. The charred cellulose granules were preliminarily steeped in ionic (NaOH, NaCl, KOH, KCl, and sodium dodecyl sulfate (SDS)), nonionic (glucose, sucrose, and ethanol), and amphipathic sucrose fatty acid ester (SFAE) solutions, and adsorption tests on the dye to the steeped and charred cellulose granules were conducted. Almost none of the dye was adsorbed when the solutions of ionic and amphipathic molecules were used, but were adsorbed in the case of steeping in the nonionic molecule solutions. Thin-layer chromatography (TLC) and the Fourier transform infra-red (FT-IR) profiles of SFAE which was adsorbed to the charred cellulose granules and extracted by ethyl ether suggested the presence of hydrophobic sites on the surface of the charred cellulose granules. We confirmed that the xanthene dyes could bind to the charred cellulose granules by ionic and hydrophobic bonds.     

Heterogeneity and Metachromasy of Some Commercial Anionic Dyes

The multicomponent character of all commercial anionic dyes tested (monoazo, disazo, indigoid and xanthene) was demonstrated by paper chromatography. On the basis of a reaction on filter paper, certain fractionated components of the dyes: aniline blue WS, benzoazurin, Bordeaux red, Congo red, cotton blue, chromotrope 2R, indigo-carmine, methyl-blau, soluble blue, and wasserblau showed a metachromatic response with the chromotropes, protamine and hexammine cobaltic chloride. The response of these same dye components with the chromotropes neomycin, polymyxin and viomycin was much weaker, and the alkaloids strychnine, codeine and cinchonidine could not elicit any metachromatic response. The hex-amminocobalt complex was the most effective of all the chromotropes studied, including protamine, both on filter paper and in aqueous solutions. Changes in color exhibited by the unchromatographed whole dyes such as alkali blue, alkali blue 6B, azoblau, Congo rubin, Hickson purple, isamine blue, orange G and trypan blue appear to be merely polychromatic effects because comparable changes are not shown by any of their chromatographically resolved components. In a solution system, the blue dyes, benzoazurin, cotton blue, indigo-carmine, methylblau, soluble blue, and wasserblau did not show definite visual changes in hue or in spectral shifts except with the hexamminocobalt complex, which induced a remarkable change in hue of all these dyes to a blue-violet or purple shade. A spectrophotometric study of methylblau has indicated that this change in hue is associated with a 25 mp shift of absorbance maximum to a lower wave length (hypsochromic effect). The filter-paper reaction between a dye component and a chromotrope is quite reliable and convenient for ascertaining a metachromatic response, since, unlike a reaction in solution systems, it is not affected by the unbound components of a reaction mixture. It is usable because water does not play any significant role in the metachromasy of anionic dyes. No correlation has been established between metachromasy and chemical constitution of anionic dyes.     

Lipid-mediated regulation of pore-forming activity of syringomycin E by thyroid hormones and xanthene dyes

The effects of dipole modifiers, thyroid hormones (thyroxine and triiodothyronine) and xanthene dyes (Rose Bengal, phloxineB, erythrosin, eosinY and fluorescein) on the pore-forming activity of the lipopeptide syringomycin E (SRE) produced by Pseudomonas syringae were studied in a model bilayer. Thyroxine does not noticeably influence the steady-state number of open SRE channels (N_op), whereas triiodothyronine decreases it 10-fold at − 50 mV. Rose Bengal, phloxine B and erythrosin significantly increase N_op by 350, 100 and 70 times, respectively. Eosin Y and fluorescein do not practically affect the pore-forming activity of SRE. Recently, we showed that hormones decrease the dipole potential of lipid bilayers by approximately 60 mV at 50 μM, while Rose Bengal, phloxine B and erythrosin at 2.5 μM reduce the membrane dipole potential by 120, 80 and 50 mV, respectively. In the present study using differential scanning microcalorimetry, confocal fluorescence microscopy, the calcein release technique and measurements of membrane curvature elasticity, we show that triiodothyronine strongly affects the fluidity of model membranes: its addition leads to a significant decrease in the temperature and cooperativity of the main phase transition of DPPC, calcein leakage from DOPC vesicles, fluidization of solid domains in DOPC/DPPC liposomes, and promotion of lipid curvature stress. Thyroxine exerts a weaker effect. Xanthene dyes do not influence the phase transition of DPPC. Despite the decrease in the dipole potential, thyroid hormones modulate SRE channels predominantly via the elastic properties of the membrane, whereas the xanthene dyes Rose Bengal, phloxine B and erythrosine affect SRE channels via bilayer electrostatics.     

Mutagenicity test of dyes used in cosmetics with the Salmonella/mammalian-microsome test.

37 dyes including 3 anthraquinone, 22 azo; 5 xanthene, 5 fluorandiol, and 2 thioindigo dyes, were tested for mutagenic potential with the Salmonella/mammalian-microsome test. Two frame-shift histidine mutants (TA1537 and TA98) and two base-pair substituted histidine mutants (TA1535 and TA100) of Salmonella typhimurium were employed. Both the spot test and the plate-incorporation assay indicated that one azo dye, D&C Orange No. 17, was mutagenic with three of the bacterial test strains. The mutagenic response of D&C Orange No. 17 was depressed by the addition of the microsomal fractions from rat livers. Of the chemicals used to synthesize D&C Orange No; 17 was depressed by the addition of the microsomal fractions from rat livers. Of the chemicals used to synthesize D&C Orange No. 17, beta-naphthol was not mutagenic but 2,4-dinitroaniline was mutagenic to the same Salmonella strains as D&C Orange No. 17 . Dimethyl sulfoxide extracts of lipsticks of similar formula but without D&C Orange No. 17 were tested in the plate incorporation assay. Only those containing D&C Orange No. 17 were mutagenic and the dye was mutagenic at concentrations consumed in normal daily use.