ALKALINE RIBONUCLEASE ACTIVITY INCREASE IN RAT KIDNEY CORTEX AND LIVER AFTER TRYPAN BLUE AND OTHER AZO DYES ADMINISTRATION

Acid azo dyes, most of them naphtholdisulfonic acid derivatives, were given intraperitoneally to rats and their effect on "alkaline" ribonuclease activity was studied in total homogenates of kidney cortex and liver. Acid treatment was used to release bound enzyme activity. Several of the dyes, including trypan blue, increased RNase activity in both organs 3 days after administration of single doses, while others, like Evans blue, were inactive. Activity was apparently bound to the sulfonic substitution in the 3, 6 positions in the naphthalene rings, substitutions in the benzidine rings being not critical. All of the active and most of the inactive compounds were taken up by tubule cells of kidney cortex and by reticular and parenchymal cells of liver. While the effect on both liver and kidney was obtained 1 day after trypan blue administration, RNase remained increased for only about 3 days in the first organ, and for at least a month in the second. However, repeated trypan blue doses increased liver enzyme activity for at least 9 days. Serum RNase activity was decreased after trypan blue administration. Ethionine administration together with trypan blue markedly blocked the effect of the dye on liver RNase activity; simultaneously given methionine partially reversed the action of the antimetabolite. This suggests that de novo synthesis of RNase is induced in liver by trypan blue. The action of ethionine on the kidney RNase response to trypan blue was less marked although significant; in view of the possible kidney uptake of the plasma enzyme, interpretation of this finding must be postponed. Results are discussed with reference to the mechanism of the structural specificity of the compounds used, cytological localization of the dyes and their mechanism of action on liver and kidney RNase.     

Staining Reactions of some Anionic Disazo Dyes and Histochemical Properties of the Red Impurity in Trypan Blue

Some staining properties of 10 anionic disazo dyes are clarified by comparison with previous chromatographic analysis. Trypan blue contains both blue and red components and the purified blue fraction displays no color shifts in tissue sections. Evans blue, Niagara blue 2B, Niagara sky blue, Niagara sky blue 4B and Niagara sky blue 6B generally resemble trypan blue. Congo red is a metachromatic dye and the only known example among anionic dyes of established purity whose color shows shifts in tissue sections and also in solutions with certain basic compounds. Other red dyes (Congo corinth, trypan red and vital red) are not metachromatic. The red dye impurity of trypan blue selectively stains nuclei which are pycnotic, degenerating or undergoing no further division. This reaction is apparently related to basic protein content. Other reactions of the red fraction of trypan blue (mammalian erythrocytes, blood plasma) are not fully explained on this basis.     

Method of counterstaining preparations for immunofluorescent studies of the pituitary]

To elucidate nonfluorescent structural elements of the hypophyseal parenchyma for immunofluorescent investigations, properties of some dyes most commonly applied for hypophysis staining have been studied. Such dyes as paraldehide-fuchsin, light green, orange G, chromotrop 2R, hematoxylin, eosin, fuchsin, azocarmin possess their own intensive luminescence and block immunofluorescence completely. Some other dyes (trypan blue, bromthymol blue, aniline blue, malachite green, methyl green) though not blocking immunofluorescence, they do not reveal hypophyseal cellular elements distinctly enough. Good results have been obtained with 0.3% water solution of toluidine blue, 0.5% solution of methylene light blue, methylene blue, as well as with Gram--Weigert's staining and with gallocyanin after Einarson. For special staining of corticotropocytes, the authors recommend 0.1% solution of bromphenol blue in barate buffer, pH 8.2.     

一种鉴定多糖水解酶类及其产生菌的新方法

利用曲里苯蓝能与多糖形成复合物的原理,建立起一种简便、快捷、灵敏的筛选多糖水解酶类及其产生菌的平板鉴定方法。曲里苯蓝对供试微生物无毒害作用,不影响酶的活性,并可高温灭菌。其最适浓度在0.005%~0.01%(W/V)之间。可用于纤维素水解酶类、淀粉水解酶类、甘露聚糖酶、普鲁兰酶等多糖水解酶,与传统方法相比,该方法不仅有着同样高的灵敏度,而且能够提高筛选效率,避免污染问题,同时适合于多种多糖底物。但不能用于木聚糖酶和菊粉酶的检测。     

dye (arc) Mutants: insights into an unexplained phenotype and its suppression by the synthesis of poly (3-hydroxybutyrate) in Escherichia coli recombinants

arcA codes for a central regulator in Escherichia coli that responds to redox conditions of growth. Mutations in this gene, originally named dye, confer sensitivity to toluidine blue and other redox dyes. However, the molecular basis for the dye-sensitive phenotype has not been elucidated. In this work, we show that toluidine blue redirects electrons to O2 and causes an increase in the generation of reactive O2 species (ROS). We also demonstrate that synthesis of poly (3-hydroxybutyrate) suppresses the Dye phenotype in E. coli recombinants, as the capacity to synthesize the polymer reduces sensitivity to toluidine blue, O2 consumption and ROS production levels.     

A comparative study of the inhibitory effect of trypan blue on mouse erythrocyte and C3 binding receptors of peripheral blood mononuclear cells from healthy donors and patients suffering from chronic lymphocytic leukaemia

Trypan blue has previously been shown to interact with the C3 receptor, but not with the Fc receptor. In the present work the effects of Trypan blue on mouse erythrocyte (ME) and C3 binding receptors of the peripheral blood mononuclear cells have been studied in healthy individuals and in patients with chronic lymphocytic leukemia (CLL). The resulting dose-response curves have been compared with each another. The results of statistical analysis indicate that there is no significant difference between the effects of trypan blue on the two receptors in healthy individuals. In contrast ME and C3 binding receptors differ significantly in trypan blue sensitivity in CLL patients. These data suggest that the two receptors are similar, or are situated on the cell membrane in such a way that the trypan blue bound to one of them inhibits the functioning of the other one too, but they are not the same. In the course of leukemic transformation, the trypan blue sensitivity of the ME binding receptor does not vary, whereas that of the C3 receptor is enhanced significantly.     

Trypan blue in vivo stains nigral dopaminergic neurons killed by 6-hydroxydopamine

Dopaminergic neurons in the substantia nigra killed by 6-hydroxydopamine were stained in vivo by intracerebral injections of trypan blue. Such staining appeared specific for dead neurons, although a proportion of these retained the ability to stain with Nissl dyes for at least 2 days. Neurons retained trypan blue in vivo for periods of up to 9 days. Trypan blue staining of some neurons outside the substantia nigra demonstrated the use of this dye in determining the degree of non-specific toxicity of 6-hydroxydopamine. Twenty-four hours after infusion of trypan blue almost no background staining was present and individually stained neurons were clearly visible. Thus the use of trypan blue may have a general application as a sensitive method for estimating discrete areas of toxin-induced neuronal death, and for estimating the degree of specificity of a toxin.     

Trypan blue in vivo stains nigral dopaminergic neurons killed by 6-hydroxydopamine

Summary Dopaminergic neurons in the substantia nigra killed by 6-hydroxydopamine were stained in vivo by intracerebral injections of trypan blue. Such staining appeared specific for dead neurons, although a proportion of these retained the ability to stain with Nissl dyes for at least 2 days. Neurons retained trypan blue in vivo for periods of up to 9 days. Trypan blue staining of some neurons outside the substantia nigra demonstrated the use of this dye in determining the degree of non-specific toxicity of 6-hydroxydopamine. Twenty-four hours after infusion of trypan blue almost no background staining was present and individually stained neurons were clearly visible. Thus the use of trypan blue may have a general application as a sensitive method for estimating discrete areas of toxin-induced neulonal death, and for estimating the degree of specificity of autoxin.     

Mutagenicity of selected sulfonated azo dyes in the Salmonella/microsome assay: use of aerobic and anaerobic activation procedures

A selection of 16 sulfonated azo dyes of both the monoazo type and diazo dyes based on benzidine, o-tolidine and o-dianisidine were assayed for mutagenicity in Salmonella typhimurium strains TA98 and TA100 employing both aerobic and anaerobic preincubation procedures. 3 food dyes, FD & C Red No. 40 and Yellows No. 5 and No. 6 were non-mutagenic in all tests. 5 dyes were mutagenic with aerobic treatment (trypan blue, Pontacyl Sky Blue 4BX, Congo Red, Eriochrome Blue Black B, dimethylaminoazobenzene) and 6 were mutagenic aerobically with riboflavin and cofactors (Deltapurpurin, trypan blue, Pontacyl Sky Blue 4BX, Congo Red, methyl orange, Ponceau 3R). Anaerobic preincubation involving enzymatic reduction of the dyes led to a different pattern of mutagenicity, with trypan blue giving much enhanced mutagenicity; Eriochrome Blue Black B, Pontacyl Sky Blue 4BX, Deltapurpurin and Congo Red exhibiting similar activity to aerobic preincubation; and methyl orange and Ponceau 3R yielding no mutagenicity. The results are interpreted with respect to an hypothesis involving partial reduction of the azo bond under differing degrees of aerobiosis via azo-anion radicals and hydrazo intermediates.     

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.     

Dead cell counts during serum cultivation are underestimated by the fluorescent live/dead assay

The live/dead fluorescent assay provides a quick method for assessing the proportion of live and dead cells in cell culture systems or tissues and is widely used. Dead cells are detected by the fluorescence produced when propidium iodide (PI) binds to DNA; PI and similar molecules are excluded from live cells but can penetrate dead cells because of their loss of membrane integrity. Here we investigated the effect of serum in the culture medium on the reliability of the method. We assessed viability of chondrocytes with/without serum using both a live/dead assay kit and also trypan blue staining. We found that after 2 days of culture, the DNA-binding dye PI could no longer detect dead cells if serum was present but they were readily detected in serum-free medium or if an inhibitor to DNase I was added to the serum-containing medium. Dead cells could be detected by trypan blue staining in all cultures. Hence dead cells are no longer detected as the DNase I present in serum degrades their DNA. DNA-binding dyes may thus not give a reliable estimate of the number of dead cells in systems that have been cultured in the presence of serum for several days.     

Chlorazol Fast Pink B And Trypan Blue Tests for Virus and Other Proteinaceous Inclusions

A 1% solution of chlorazol fast pink B in 0.9% NaCl can be used like trypan blue to detect virus inclusions and proteinaceous entities in peelings from leaves or thin sections taken from living plant tissue. Like trypan blue, a solution of the pink dye causes somatic nuclei to swell and thus facilitates observation of their structure. The two dyes combine into a beautiful differential bicolored stain. Mix 5 ml of 0.5% trypan blue stock solution with 35 ml of 1% chlorazol pink B in 0.9% NaCl. Stain fresh tissue 1-2 minutes. The combination stain is superior to either dye alone for differentiating virus entities.     

Mutagenicity of benzidine and benzidine-congener dyes and selected monoazo dyes in a modified Salmonella assay

We have evaluated the mutagenic activity of a series of diazo compounds derived from benzidine and its congeners o-tolidine, o-dianisidine and 3,3'-dichlorobenzidine as well as several monoazo compounds. The test system used was a modification of the standard Ames Salmonella assay in which FMN, hamster liver S9 and a preincubation step are used to facilitate azo reduction and detection of the resulting mutagenic aromatic amines. All of the benzidine and o-tolidine dyes tested were clearly mutagenic. The o-dianisidine dyes except for Direct Blue 218 were also mutagenic. Direct Blue 218 is a copper complex of the mutagenic o-dianisidine dye Direct Blue 15. Pigment Yellow 12, which is derived from 3,3'-dichlorobenzidine, could not be detected as mutagenic, presumably because of its lack of solubility in the test reaction mixture. Of the monoazo dyes tested, methyl orange was clearly mutagenic, while C.I. Acid Red 26 and Acid Dye (C.I. 16155; often referred to as Ponceau 3R) had marginal to weak mutagenic activity. Several commercial dye samples had greater mutagenic activity with the modified test protocol than did equimolar quantities of their mutagenic aromatic amine reduction products. Investigation of this phenomenon for Direct Black 38 and trypan blue showed that it was due to the presence of mutagenic impurities in these samples. The modified method used appears to be suitable for testing the mutagenicity of azo dyes, and it may also be useful for monitoring the presence of mutagenic or potentially carcinogenic impurities in otherwise nonmutagenic azo dyes.     

Dyes,trypanosomiasis and DNA: a historical and critical review

Abstract

Trypanosomiasis, a group of diseases including sleeping sickness in humans and Nagana in cattle in Africa, and Chagas’ disease in South America, remains a considerable problem in the 21^st century. The therapies that are available, however, usually have their roots in the “dye therapy” of a century ago, knowledge gained at the microscope from parasite staining procedures and converted to chemotherapy based on compounds closely related to the laboratory reagents. Dyes such as trypan red and trypan blue led to the development of suramin, while cationic nitrogen heterocyclic dyes furnished examples of the phenanthridinium class, such as ethidium (homidium) and isometamidium. Both suramin and isometamidium remain in use. Owing to mutagenicity issues, the presence of ethidium among the phenanthridinium dyes has led to concerns over the clinical use of related derivatives. There are several mechanisms for dye-DNA interaction, however, including possible hydrogen bonding of dye to the polymer, and these are discussed together with structure-activity relations and cellular localization of the phenanthridine and isomeric acridines involved. Better understanding of nucleic acid binding properties has allowed the preparation of more effective phenanthridinium analogues intended for use as anticancer/antiviral therapy.     

伊红、台盼蓝检测河蟹精子存活率的比较

对台盼蓝和伊红染色法检测河蟹(Eriocheir sinensis)精子存活率的方法进行了评价研究。结果表明,两种染色法死、活精子分别呈现出明显不同的染色特征:活精子无色透明,顶体中央凸起呈圆锥状,光镜下辐射臂及细胞边界清晰;死亡精子顶体着色,且中央有一染色较深的圆斑,核杯染色不明显,细胞体积变大,边界模糊。通过不同染色时间和不同染料浓度的比较发现,两种染色法最适染液浓度分别是0·25%的伊红和0·5%的台盼蓝,染色时间均以15min为佳。在此基础上,将新鲜精子和60℃水浴处理致死精子以不同的体积比混合,配成含致死精子比例为10%～90%的9个梯度样品,用伊红和台盼蓝分别测定各样品精子死亡率,并进行相关性分析。结果发现,各样品实测精子死亡率均略高于样品的理论死亡率,同时两种染色法实测值与样品理论值呈显著正相关(P<0·05),两种染色法之间亦呈显著正相关(P<0·05)。上述结果表明,伊红和台盼蓝可用于河蟹精子的活体染色,且两种染色法在对河蟹精子染色中具有一定的稳定性和可比性。     

The trypanocidal drug suramin and other trypan blue mimetics are inhibitors of pyruvate kinases and bind to the adenosine site

Ehrlich's pioneering chemotherapeutic experiments published in 1904 (Ehrlich, P., and Shiga, K. (1904) Berlin Klin. Wochenschrift 20, 329-362) described the efficacy of a series of dye molecules including trypan blue and trypan red to eliminate trypanosome infections in mice. The molecular structures of the dyes provided a starting point for the synthesis of suramin, which was developed and used as a trypanocidal drug in 1916 and is still in clinical use. Despite the biological importance of these dye-like molecules, the mode of action on trypanosomes has remained elusive. Here we present crystal structures of suramin and three related dyes in complex with pyruvate kinases from Leishmania mexicana or from Trypanosoma cruzi. The phenyl sulfonate groups of all four molecules (suramin, Ponceau S, acid blue 80, and benzothiazole-2,5-disulfonic acid) bind in the position of ADP/ATP at the active sites of the pyruvate kinases (PYKs). The binding positions in the two different trypanosomatid PYKs are nearly identical. We show that suramin competitively inhibits PYKs from humans (muscle, tumor, and liver isoenzymes, K(i) = 1.1-17 μM), T. cruzi (K(i) = 108 μM), and L. mexicana (K(i) = 116 μM), all of which have similar active sites. Synergistic effects were observed when examining suramin inhibition in the presence of an allosteric effector molecule, whereby IC(50) values decreased up to 2-fold for both trypanosomatid and human PYKs. These kinetic and structural analyses provide insight into the promiscuous inhibition observed for suramin and into the mode of action of the dye-like molecules used in Ehrlich's original experiments.     

Redox-mediated decolorization of recalcitrant textile dyes by <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> WL1 laccase

The efficiency of crude and partially purified Trichoderma harzianum WL1 laccase for the decolorization of synthetic dyes (Rhodamine 6G, Erioglaucine and Trypan blue) with complex aromatic structures were evaluated. Selection of dyes was based on their extensive usage in local dyeing and textile industries around the study area. Studies on the role of redox potential of laccases on dye decolorization are rarely discussed and hence, for the first time we have shown the redox mediated dye decolorizing efficiency of T. harzianum WL1 laccase with the commonly employed redox mediator 1-hydroxybenzotriazole (HBT). The process parameters such as initial dye concentration, enzyme load and HBT concentration were studied and found that they had a great influence on dye removal process. When the dyes were treated with increased concentration of enzyme, it showed a greater percentage of decolorization. Compared to the crude laccase, partially purified laccase accounts for maximum decolorization of all the dyes studied. In addition, the rate of dye decolorization was considerably enhanced in presence of 4 mM HBT. Maximum and minimum decolorization were recorded for Rhodamine 6G and Trypan blue, respectively. The results of this study further confirmed that, T. harzianum laccase was found to be suitable with HBT and this laccase-mediator system (LMS) could be applied for the decolorization of various classes of dyes.     

The use of vital dyes to assess embryonic viability in the hamster, Mesocricetus auratus

Experiments were designed to assess the use of the vital dyes trypan blue and fluorescein diacetate as indicators of the viability of hamster ova and embryos. Exclusion of trypan blue and fluorescence with fluorescein diacetate showed high correlations with uptake of [3H]uridine by ova and further development of embryos in vitro. Ova killed by freezing and thawing incorporated [3H]uridine at background levels. Trypan blue exclusion and fluorescein diacetate uptake were highly correlated with each other (r = 0.99). Trypan blue and fluorescein diacetate serve as excellent indices of viability in ova and early embryos of hamsters.     

The role of sulphur, sulphide and reducible dyes in the enzymic oxidation of cysteamine to hypotaurine

1. Cysteamine is oxidized to hypotaurine by an enzyme extracted from horse kidney, with sulphur or sulphide acting as a cofactor. It has been now found that, when the enzyme is omitted, sulphur and sulphide are able to catalyse the oxidation of cysteamine to cystamine by molecular oxygen. 2. Methylene blue may be used in catalytic amounts as a cofactor in the enzymic oxidation of cysteamine to hypotaurine in the place of sulphur or sulphide. The effect of methylene blue is not light-dependent and is not abolished by catalase. Other redox dyes with E'(0) higher than that of methylene blue are also used as cofactors. 3. A property common to all the cofactors is that they are necessary for the enzymic process in catalytic amounts, though they depress the final amount of hypotaurine produced when added over a critical concentration. All the cofactors share also the property of being catalysts for the non-enzymic oxidation of cysteamine to cystamine. 4. Methylene blue is reduced by cysteamine under anaerobic conditions, and is reoxidized in the presence of air. The rate of the reduction is not accelerated by the enzyme, indicating that the dye does not act in this reaction as a hydrogen carrier from the enzyme to oxygen. The possible mechanism of action of methylene blue and of the other cofactors is discussed.     

Horseradish peroxidase catalyzed degradation of industrially important dyes.

Horseradish peroxidase (HRP) is known to degrade certain recalcitrant organic compounds such as phenol and substituted phenols. Here, for the first time we have shown HRP to be effective in degrading and precipitating industrially important azo dyes. For Remazol blue, the enzyme activity was found to be far better at pH 2.5 than at neutral pH. In addition, Remazol blue acts as a strong competitive inhibitor of HRP at neutral pH. Horseradish peroxidase shows broad substrate specificity toward a variety of azo dyes. Kinetic constants (K(m)(app) and V(max)(app)) for two different dyes have been determined. In addition to providing a systematic analysis of the potential of HRP in degradation of dyes, this study opens up a new area on exploration of commercial dyes as inhibitors of enzymes. 2001 John Wiley & Sons, Inc.