Interaction of ethidium bromide with DNA as studied by kinetic spectrophotometry.

The interaction of ethidium bromide (EB) with DNA has been investigated using the pulse radiolysis technique. In particular, the absolute rate constant for the reaction of hydrated electrons, generated by single pulses of high-energy electrons, with EB is shown to drop dramatically in the presence of DNA. This drop in diffusion-limited reactivity results from the interaction of EB with DNA, effectively immobilising it, thus lowering the reaction cross-section or probability. Analysis of the resulting kinetic spectrophotometric data shows that they are consistent with a reversible interaction of EB with DNA as described by the law of mass action. The Scatchard-type plots obtained are linear, and give quantitative information on the extent and degree of association, comparable with that obtained by more conventional methods. The potential of the pulse radiolysis technique for studying different types of interactions between small molecules and various biopolymers has been demonstrated.     

Effects of ethidium bromide and berenil on protein synthesis

The effects of ethidium bromide, an intercalating dye and berenil, a nonintercalating dye on the biological activities ofEscherichia coli ribosomes have been studied. Ethidium bromide treatment drastically reduced both enzymatic and nonenzymatic initiation complex formation, enzymatic as well as nonenzymatic binding of phenylalanyl tRNA, peptidyl transferase, GTPase as well as the overall protein synthesising activity as measured by the poly U-dependent polymerization of phenylalanine. On berenil treatment, however, only enzymatic formation of the initiation complex is marginally reduced. Other reactions are not markedly affected except the enzymatic phenylalanyl tRNA binding which is slightly decreased only at high Mg²⁺ concentration; the treated ribosome has lowered polymerizing activity at sub-optimal Mg²⁺ concentration (10 mM). Although it has already been shown in this laboratory that treatment with either dye leads to the unfolding of the structure of the ribosome, the present studies indicate that berenil treatment does not alter the structure of the ribosome drastically in contrast to ethidium bromide treatment.     

Highly Selective Acridine and Ethidium Staining of Bacterial DNA and RNA

The acridine dyes acridine orange (AO) and coriphosphine O (CPO) and ethidium bromide (EtBr) were used to stain bacterial digests after electrophoresis in native and denaturing (SDS) polyacrylamide gels and were shown to stain DNA and RNA preferentially over other subcellular components in the gels. Vegetative cell digests of Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus showed intense staining of DNA with AO and CPO near the top of the gel, but little or no staining of other cellular constituents. EtBr stained both DNA and RNA in the gels. Protein standards and non-nucleic acid cellular constituents stained faintly with high concentrations (> 100 μM) of AO, lower concentrations (13.9 μM) of CPO, and did not stain with 0.5 μg/ml EtBr in denaturing gels. The complete set of cellular biochemicals was visualized by silver staining, while the protein subset was detected by Coomassie blue staining. The highest concentrations of AO (120 μM) and CPO (13.9 μM) were shown to detect purified DNA in gels with a sensitivity in the range of 25–50 ng per band. This work demonstrates the specificity of acridine and ethidium dyes for nucleic acids, while illustrating the level of non-nucleic acid-specific interactions with other cellular components by staining of electrophoretically separated cellular components in a gel matrix.     

Propidium: induction of petites and recovery from ethidium mutagenesis in Saccharomyces cerevisiae.

It was shown that petite induction in growing cells of $\text{Saccharomyces}$$\text{cerevisiae}$ by ethidium was strongly stimulated by the presence of propidium, a phenanthridinium dye of similar structure to ethidium. Propidium itself also induced petites in growing but not in resting cells. Furthermore, propidium could prevent petite induction in resting cells and caused recovery from ethidium induction with prolonged incubation. A possible mode of action of propidium in the ethidium-induced petite mutagenesis is discussed.     

Ethidium bromide enhancement of frameshift mutagenesis caused by photoactivatable ethidium analogs.

Ethidium azide analogs (3-amino-8-azido-ethidium monoazide and ethidium diazide) have been developed as photosensitive probes in order to analyze directly the reversible in vivo interactions of ethidium bromide. Our preliminary observations [11], relating the mutagenic potential of the monoazide analog of ethidium, have been extended and refined, using the highly purified ethidium azide analogs [5]. A number of physical-chemical studies indicate that the monoazide analog interaction with nucleic acids, prior to photolysis, resembles remarkably the interaction of the parent ethidium (unpublished). It was anticipated, therefore, that competition by ethidium for the ethidium monoazide mutagenic sites in Salmonella TA1538 would be observed when these drugs were used in combination. Previous results in fact showed a decreased production of frameshift mutants when ethidium bromide was added to the ethidium monoazide in the Ames assay [1]. However, more extensive investigations, reported here, have shown that this apparent competition was the result of neglecting the toxic effects of ethidium monoazide and its enhanced toxocity in the presence of ethidium bromide. Conversely, an enhancement of the azide mutagenesis and toxicity for both the mono- and diazide analogs was seen when ethidium bromide was used in combination with these analogs.     

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.     

溴化乙锭诱导无线粒体DNA宫颈癌HeLa细胞系的构建和鉴定

[目的]构建和鉴定由溴化乙锭(EB)诱导的无线粒体DNA(mtDNA)宫颈癌ρ~0HeLa细胞系,探讨mtDNA与宫颈癌发生的关系。[方法]采用含50ng/ml溴化乙锭、100μg/ml丙酮酸钠和50μg/ml尿嘧啶核苷的高糖DMEM完全培养基中传代培养HeLa细胞。低剂量EB连续诱导60d后,采用营养缺陷鉴定、PCR和WesternBlot鉴定无mtDNA的ρ~0HeLa细胞系;采用透射电子显微镜观察ρ~0HeLa细胞内线粒体形态变化;采用CCK8法测定ρ~0HeLa细胞增殖曲线。[结果]经溴化乙锭诱导60d,可以培养出具有尿嘧啶核苷依赖性的无mtDNA宫颈癌HeLa细胞系。普通PCR和qPCR结果均显示,低剂量EB诱导60d的ρ~0HeLa细胞中mtDNA完全缺失。WesternBlot结果显示,HeLa细胞中能表达核编码的NDUFA9蛋白,也能表达线粒体编码MT-ND1蛋白。而ρ~0HeLa细胞中已无MT-ND1蛋白表达,但核编码的NDUFA9蛋白能够正常表达。透射电子显微镜观察显示,ρ~0HeLa细胞内部分出现空泡改变,线粒体嵴被破坏。CCK8细胞增殖实验结果显示,ρ~0HeLa细胞系生长速度显著低于正常HeLa细胞系,且差异有统计学意义(P<0.05)。[结论]无mtDNA的宫颈癌HeLa细胞系的建立,为后续研究mtDNA突变和线粒体功能在宫颈癌发生发展中的作用及机制奠定了基础。     

Effects of ethidium bromide and chloramphenicol on the mitochondrial nucleoids in Euglena gracilis

The effects of ethidium bromide (EB) at 0.13 m M and of chloramphenicol (CAP) at 46 m M on the mitochondria and mitochondrial nucleoids in Euglena gracilis . Z strain, were examined by fluorescence microscopy and by electron microscopy. Ethidium bromide stopped the multiplication of cells and decreased their respiratory activity by 55% after treatment for 10 days. Most of the mitochondria became slender with few cristae and some became cup-shaped with stacked cristac. Mitochondrial nucleoids decreased markedly in number after treatment with EB for more than 2 days. After treatment for 3 days with EB, mitochondrial nucleoids could not be detected in about half of all cells examined. Treatment with CAP for 10 days reduced the respiratory activity by 47%. Chloramphenicol did not decrease the number of mitochondrial nucleoids but it increased the number of cristae and the volume of mitochondria.     

Adsorption of ethidium bromide from aqueous solution onto nutraceutical industrial fennel seed spent: Kinetics and thermodynamics modeling studies

Dye pollutants from research laboratories are one of the major sources for environmental contamination. In the present study, a nutraceutical industrial fennel seed spent (NIFSS) was explored as potential adsorbent for removal of ethidium bromide (EtBr) from aqueous solution. The adsorbent was characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Through batch experiments, the operating variables like initial dye concentration, adsorbent dosage, temperature, contact time, and pH were optimized. Equilibrium data were analyzed using three number of two-parameter and six number of three-parameter isotherm models. The adsorption kinetics was studied using pseudo-first order and pseudo-second order. The diffusion effects were studied by film diffusion, Webber–Morris, and Dumwald–Wagner diffusion models. The thermodynamic parameters; change in enthalpy (ΔHº), entropy (ΔSº), and Gibbs free energy (ΔGº) of adsorption system were also determined and evaluated.     

Modulation of DNA intercalation by resveratrol and genistein

Time correlated Single Photon Counting study (TCSPC) was performed for the first time to evaluate the effect of resveratrol (RES) and genistein (GEN) at 10–100 μM and 10–150 μM respectively, in modulating the DNA conformation and the variation induced due to intercalation by the dyes, ethidium bromide (EtBr) and acridine orange (AO). It is demonstrated using UV-absorption and fluorescence spectroscopy that RES and GEN, at 50 μM and 100 μM respectively can bind to DNA resulting in significant de-intercalation of the dyes, preventing their further intercalation within DNA. Hyperchromicity with red/blue shifts in DNA when bound to dyes was reduced upon addition of RES and GEN. DNA-dependent fluorescence of EtBr and AO was quenched in the presence of RES by 87.97% and 79.13% respectively, while similar quenching effect was observed for these when interacted with GEN (85.52% and 83.85%). It is found from TCSPC analysis that the higher lifetime component or constituent of intercalated dyes (τ₂, A ₂) decreased with the subsequent increase in smaller component or constituent of free dye (τ₁, A ₁) after the interaction of drugs with the intercalated DNA. Thus these findings signify that RES and GEN can play an important role in modulating DNA intercalation, leading to the reduction in DNA-directed toxicity.     

Fate during cell growth of yeast mitochondrial and nuclear DNA after photolytic attachment of the monoazide analog of ethidium.

The ¹⁴C-labeled photosensitive monoazide analog of ethidium, 3-amino-8-azido-5-ethyl-6-phenylphenanthridinium chloride, produced covalent adducts in yeast cells with both nuclear and mitochondrial DNA on photolysis by visible light. With subsequent cultivation in nutrient medium, drug molecules on mitochondrial DNA were removed only through extensive mitochondrial DNA degradation. In contrast, drug attached to nuclear DNA was eliminated with conservation of DNA, presumably through a repair process.     

Micronomicin/tobramycin binding with DNA: fluorescence studies using of ethidium bromide as a probe and molecular docking analysis

Two aminoglycosides, micronomicin (MN), and tobramycin (TB), binding with DNA were studied using various spectroscopic techniques including fluorescence, UV–Vis, FT-IR, and CD spectroscopy coupled with relative viscosity and molecular docking. Studies of fluorescence quenching and time-resolved fluorescence spectroscopy all revealed that MN/TB quenching the fluorescence of DNA–EB belonged to static quenching. The binding constants and binding sites were obtained. The values of ΔH, ΔS, and ΔG suggested that van der Waals force or hydrogen bond might be the main binding force. FT-IR and CD spectroscopy revealed that the binding of MN/TB with DNA had an effect on the secondary structure of DNA. Binding mode of MN/TB with DNA was groove binding which was ascertained by viscosity measurements, CD spectroscopy, ionic strength, melting temperature (T_m), contrast experiments with single stranded (ssDNA), and double stranded DNA (dsDNA). Molecular docking analysis further confirmed that the groove binding was more acceptable result.     

Investigation of binding properties of two ethidium derivatives with serum albumins: spectral and computational approach

The interaction mechanisms of two ethidium derivatives, 3,8-dibenzoylamino-5-ethyl-6-phenylphenantridinium chloride (E2) and 3,8-diphenylacetylamino-5-ethyl-6-phenylphenantridinium chloride (E3) with serum albumins (BSA and HSA) have been investigated by a combined experimental and computational approach. Fluorescence quenching and UV–vis results revealed that the interaction of derivatives with albumins resulted in formation of ground-state complexes and the obtained Stern–Volmer quenching constants designate the presence of a static component in the quenching mechanisms. Thermodynamic parameters (ΔH and ΔS values) point out the ionic interactions play the major role in E2-BSA, E2-HSA and E3-HSA complexes. The van der Waals interactions are dominant forces in E3-BSA complex. Moreover, the obtained results in this study were supported with computational analyzes which have same tendency.     

Biogenesis of mitochondria: XXV. Studies on the mitochondrial genomes of petite mutants of yeast using ethidium bromide as a probe

This paper describes investigations into the effects of ethidium bromide on the mitochondrial genomes of a number of different petite mutants derived from one respiratory competent strain of Saccharomyces cerevisiae. It is shown that the mutagenic effects of ethidium bromide on petite mutants occur by a similar mechanism to that previously reported for the action of this dye on grande cells. The consequences of ethidium bromide action in both cases are inhibition of the replication of mitochondrial DNA, fragmentation of pre-existing mitochondrial DNA, and the induction, often in high frequency, of cells devoid of mitochondrial genetic information (ρ ° cells).The susceptibility of the mitochondrial genomes to these effects of ethidium bromide varies in the different clones studied. The inhibition of mitochondrial DNA replication requires higher concentrations of ethidium bromide in petite cells than in the parent grande strain. Furthermore, the susceptibility of mitochondrial DNA replication to inhibition by ethidium bromide varies in different petite clones.It is found that during ethidium bromide treatment of the suppressive petite clones, the over-all suppressiveness of the cultures is reduced in parallel with the reduction in the over-all cellular levels of mitochondrial DNA. Furthermore, ethidium bromide treatment of petite clones carrying mitochondrial erythromycin resistance genes (ρ^?ER^r) leads to the elimination of these genes from the cultures. The rates of elimination of these genes are different in two ρ^?ER^r clones, and in both the gene elimination rate is slower than in the parent ρ⁺ ER^r strain. It is proposed that the rate of elimination of erythromycin resistance genes by ethidium bromide is related to the absolute number of copies of these genes in different cell types. In general, the more copies of the gene in the starting cells, the slower is the rate of elimination by ethidium bromide. These concepts lead us to suggest that petite mutants provide a system for the biological purification of particular regions of yeast mitochondrial DNA and of particular relevance is the possible purification of erythromycin resistance genes.     

Binding of ampholine to transfer RNA.

The melting temperature of isoaccepting tRNAfMet is affected by Ampholine. The plot of Tm versus the logarithm of Ampholine concentration shows clearly an increasing effect of Ampholine when the pH changes from 7.4 to 4.2. This result is interpreted as binding of Ampholine to the nucleic acid. The effects of Ampholine have been compared with those of soidum, magnesium and tetraethylene pentamine. Ampholine carrier ampholytes at pH 4.2 bind to tRNA with the same affinity as magnesium; at higher pH values they are less active. An hypothesis for the mechanism of action of Ampholine on nucleic acids during isoelectric focusing is proposed.