Spectroscopic and molecular modeling studies of caffeine complexes with DNA intercalators.

Recent studies have demonstrated that caffeine can act as an antimutagen and inhibit the cytoxic and/or cytostatic effects of some DNA intercalating agents. It has been suggested that this inhibitory effect may be due to complexation of the DNA intercalator with caffeine. In this study we employ optical absorption, fluorescence, and molecular modeling techniques to probe specific interactions between caffeine and various DNA intercalators. Optical absorption and steady-state fluorescence data demonstrate complexation between caffeine and the planar DNA intercalator acridine orange. The association constant of this complex is determined to be 258.4 +/- 5.1 M-1. In contrast, solutions containing caffeine and the nonplanar DNA intercalator ethidium bromide show optical shifts and steady-state fluorescence spectra indicative of a weaker complex with an association constant of 84.5 +/- 3.5 M-1. Time-resolved fluorescence data indicate that complex formation between caffeine and acridine orange or ethidium bromide results in singlet-state lifetime increases consistent with the observed increase in the steady-state fluorescence yield. In addition, dynamic polarization data indicate that these complexes form with a 1:1 stoichiometry. Molecular modeling studies are also included to examine structural factors that may influence complexation.     

A protective effect of caffeine on the ethidium induced petite mutation in yeast

A prerequisite for petite induction by ethidium bromide (EB) is an initial covalent attachment of the drug to cytoplasmic DNA. This DNA modification is thought to initiate repair processes. The repair inhibitor, caffeine, provided a protective effect against the ethidium induced petite mutation at caffeine concentrations known to inhibit the repair of UV damage in cytoplasmic DNA (Fig. 1). Mitochondrial DNA isolated from yeast exposed to EB in vivo was not as degraded in the presence of both drugs as with EB alone (Fig. 2).     

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.     

Permeability of the membrane of the Escherichia coli envelope for ethidium bromide

The interaction of ethidium bromide, a fluorescent dye, with Escherichia coli cells was studied. The envelope of intact cells was shown to be impermeable for ethidium bromide molecules. The dye penetrated however into E. coli spheroplasts. The barrier properties of the cell envelope against ethidium bromide were ruptured if the cells were treated with EDTA. The results suggest that the outer membrane serves as a principal barrier against penetration of ethidium bromide inside the cells while the cytoplasmic membrane of E. coli is permeable for the dye.     

Evidence of altered histone interactions, as investigated by removal of histones, in chromatin isolated from rat liver nuclei by a conventional method.

It is shown that the release of the slightly lysine-rich histones f2a2 and f2b by 0.4 M ammonium sulfate from conventionally isolated chromatin is diminished in comparison to the lysed nuclei. The change in extractability is further demonstrated by the application of ethidium bromide. At a molar input ratio of 0.09 (moles ethidium bromide/moles nucleotide) and 0.4 M ammonium sulfate the slightly lysine-rich histones are released from the chromatin to 70 - 80% if the lysed nuclei are used. At 0.1 M ammonium sulfate ethidium bromide effected also a release of 50 % of histone f1. Comparable effects could not be observed with chromatin prepared in a conventional way but instead a tendency towards loss of histone f3 in the presence of ethidium bromide was observed.     

In Pseudomonas aeruginosa ethidium bromide does not induce its own degradation or the assembly of pumps involved in its efflux

Xu et al. [Biochem. Biophys. Res. Commun. 305 (2003) 941] reported that, when a mutant strain of Pseudomonas aeruginosa lacking its major multidrug efflux pump complex, MexAB-OprM, was incubated with 100 μM ethidium bromide, the fluorescence, caused by its binding to DNA following its entry into cells, decreased gradually. The authors concluded that the intracellular ethidium bromide “induced” either its degradation or its efflux through the assembly of unknown efflux pumps. We found, through quantitation of ethidium bromide by absorption spectroscopy, that the total amount of ethidium bromide in the system remained constant under these conditions, indicating the absence of its degradation. Furthermore, intracellular ethidium bromide kept increasing during the experiment, showing that the decrease of fluorescence was due to self-quenching, and that ethidium bromide is not pumped out by a newly assembled efflux system.     

Characterization of an ATP-binding cassette from Clostridium perfringens with homology to an ABC transporter from Clostridium hathewayi

A ciprofloxacin-resistant mutant of Clostridium perfringens, strain VPI-C, which had stable mutations in the topoisomerase genes, accumulated less norfloxacin and ethidium bromide than the wild type, strain VPI. Efflux pump inhibitors both increased the accumulation of ethidium bromide by cells of the mutant and enhanced their sensitivity to this toxic dye. Cloning a gene, which codes for a putative ABC transporter protein (NP_562422) of 527 amino acids, from the mutant strain VPI-C into the wild-type strain VPI not only reduced the accumulation of ethidium bromide by the recombinant strain but also reduced its sensitivity to norfloxacin and ciprofloxacin. Efflux pump inhibitors decreased the rate at which ethidium bromide was removed from the cells of the recombinant strain. It appears that the putative ABC transporter protein (NP_562422) may contribute to extrusion of drugs from C. perfringens.     

An assay for proteinases and their inhibitors based on DNA/ethidium bromide fluorescence

Proteinases and their inhibitors have become the subject of intense research interest recently, since they control a multitude of very important biological processes, from the development of lambda phage to hypertension in humans. We have developed a simple and sensitive assay for detecting the activity of proteinases and of their proteinase inhibitors. The assay is based on ethidium bromide fluorescence, according to the following principles: (i) Ethidium bromide increases its fluorescence by 25-fold when it intercalates between base pairs of double-stranded DNA. (ii) Histones prevent this large increase in fluorescence by binding with high affinity to DNA thus blocking ethidium bromide intercalation. (iii) A proteinase that digests histones will make more DNA available for ethidium bromide intercalation, thereby producing an increase of fluorescence. Proteinase activity can easily be determined, in the presence of a DNA/histone complex, from the rate of ethidium fluorescence increase. In contrast, activity of a proteinase inhibitor is quantitated by the inhibition of fluorescence gain in the presence of a known amount of proteinase. This assay is rapid, simple, inexpensive, and, at the same time, accurate and sensitive enough to allow quantitation of nanogram amounts of various broad-specificity proteinases and their inhibitors. We show some possible applications of the assay (i) in testing column fractions during protein purifications, (ii) quantitation of alpha 1-antitrypsin in human serum, and (iii) detection of proteinase activity in cell extracts.     

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.     

Circular dichroism studies of ethidium bromide binding to the isolated nucleolus.

Circular dichroism in the 300-360 nm region and fluorescence induced by intercaltating binding of ethidum bromide to both DNA and RNA components were studied in isolated HeLa nucleoli. Both DNA and RNA compoents contribute to the induced dichroic elliticity. Digestion of nucleoli by RNase or DNase shows that most of the induced ellipticity comes from the DNA component. In nucleoli with an RNA/DNA = 0.8/1.0 the RNA component gives only 20% of the total ellipticity when measured at an ethidium bromide/DNA = 0.25. Spectro-fluorometric titration shows that ethidium bromide intercalates mostly into DNA in nucleoli. Both circular dichroism and fluorescence studies indicate that both DNA and RNA components in isolated nucleoli are less accessible to intercalating binding by ethidium bromide when compared to purified nucleolar DNA, DNA in chromatin or purified ribosomal RNA. Circular dichroic measurements of intercalating binding of ethidium bromide to to nucleoli may be used to study changes in nucleoli under different physiological or pathological conditions.     

Differential effects of nalidixate on the cell growth of respiratory competent strains and cytoplasmic petite mutants of Saccharomyces cerevisiae

Summary Sodium nalidixate inhibited the cell growth and division of several respiratory competent strains of Saccharomyces cerevisiae. A number of cytoplasmic petite strains (both spontaneous and induced by ethidium bromide) were shown to be more resistant to sodium nalidixate than the wild-type strains from which they were derived. There was considerable variation in sensitivity of different petites derived from the same wild-type. Usually petite strains which were induced by ethidium bromide were more resistant than spontaneously arising petites. The susceptibility of a wild-type strain to nalidixate was found to be least when the mitochondrial respiratory system was maximally repressed. It was also noted that sodium nalidixate (100 g/ml) induced petite mutants.Dr. Carnevali is a Senior Research Worker of the Centro di Studio per gli Acidi Nucleici of the National Research Council of Rome and is on leave of absence at the above address     

A clarification of the complex spectrum observed with the ultraviolet circular dichroism of ethidium bromide bound to DNA.

Ethidium bromide intercalation strongly effects the circular dichroism spectrum of DNA in the region of 230-300 mu, in a complex manner. In this report we present a study that quantitizes the relationships of the circular dichroism spectrum in the region of 230-300 mu and the ethidium bromide induced optical activity centered around 308 mu. We present evidence of two hidden cooperative bands that are probably the negative counterparts of the 308 mu band and 330 mu shoulder positive cooperative bands. The hidden band is quantitatively characterized. We confirm that the direct effect of ethidium bromide on the DNA spectrum is simply linearly proportional to the amount of intercalated dye. We also observe that the ethidium bromide enters freely when there is a molecule intercalated for every 3 sites, but that the intercalation is more difficult when the molecule intercalates at every second site.     

Study of the DNA/ethidium bromide interactions on mica surface by atomic force microscope: influence of the surface friction

The influence of mica surface on DNA/ethidium bromide interactions is investigated by atomic force microscopy (AFM). We describe the diffusion mechanism of a DNA molecule on a mica surface by using a simple analytical model. It appears that the DNA diffusion on a mica surface is limited by the surface friction due to the counterion correlations between the divalent counterions condensed on both mica and DNA surfaces. We also study the structural changes of linear DNA adsorbed on mica upon ethidium bromide binding by AFM. It turns out that linear DNA molecules adsorbed on a mica surface are unable to relieve the topological constraint upon ethidium bromide binding. In particular, strongly adsorbed molecules tend to be highly entangled, while loosely bound DNA molecules appear more extended with very few crossovers. Adsorbed DNA molecules cannot move freely on the surface because of the surface friction. Therefore, the topological constraint increases due to the ethidium bromide binding. Moreover, we show that ethidium bromide has a lower affinity for strongly bound molecules due to the topological constraint induced by the surface friction.     

Destruction of ethidium bromide in solution by ozonolysis

Ethidium bromide can be rapidly destroyed in aqueous solutions or in isoamyl alcohol by ozonolysis in the presence of H2O2 to give a mixture of organic acids. In a variety of buffers commonly used in recombinant DNA technology destruction of ethidium bromide was more than 99.9%. The yellow reaction mixture after ozonolysis was shown to be nonmutagenic. This method may be used in laboratories for the disposal of ethidium bromide wastes.     

Genotypic and phenotypic characteristics of L-line cells resistant to ethidium bromide

Stable mutants resistant to ethidium bromide in concentrations of 1 and 3 micrograms/ml have been selected in a single step in L cells. The frequency of spontaneously occurring ethidium bromide resistant clones after the exposure to 1 microgram/ml of the drug has been established as 5.10(-5). Resistant variants were induced following treatment with mutagen N-methyl-N-nitro-N-nitrosoguanidine. The resistant clones were shown to be resistant to higher concentration of the agent then which was used for selection. In multistep selection, a number of clones resistant to ethidium bromide in concentration up to 50 micrograms/ml was obtained. The alteration in the permeability of plasma membrane to the drug is the clue mechanism of the resistance.     

Induction of respiration deficient mutants in Saccharomyces cerevisiae by Berenil

Summary Some characteristic details of mutagenesis by Berenil, a non-intercalating trypanocidal dye, that govern the change from wild type (⁺) to vegetative petite (^–) in Saccharomyces cerevisiae are presented and contrasted with the intercalating mutagens ethidium bromide and euflavine.The extent and rate of mutagenesis by Berenil is affected by a variety of parameters controlling the cellular and mitochondrial phenotype: among them are exposure to 45°; competition with EB but not euflavine; a requirement for an energy source during and subsequent to exposure to the mutagen; exposure to caffeine; and the presence of genetic blocks in various steps of the mitochondrial repair system for uv-induced lesions. It is, however, insensitive to exposure to Antimycin A. Except for the first of these observations, qualitative differences have emerged between the responses induced by Berenil and the other mutagens, especially ethidium bromide.Using these observations we have postulated a stepwise sequence of events that can account for the mutagenic action of Berenil.Publication No. 2122.     

The participation of the transport-barrier functions of the plasma membrane in the development of fluoroquinolone (ciprofloxacin) resistance in Acholeplasma laidlawii]

The role of transport activity of Acholeplasma laidlawii plasmatic membrane in the development of resistance to ciprofloxacin was investigated. It was shown that ethidium bromide used as fluoroquinolone analogue in plasmatic membrane efflux pump was accumulated in ciprofloxacin-resistant cells in much less amount. It was estimated that ethidium bromide efflux depended on temperature, glucose and transmembrane electro-chemical proton potential. Inhibitors of efflux systems--reserpine and verapamil enhanced the ethidium bromide accumulation much more intensively in ciprofloxacin resistant cells. The results of investigation allowed to consider the existence of active efflux system for toxic agents in acholeplasma; in the case of ciprofloxacin-resistant strain these systems are inducible.     

Hydration effects accompanying the formation of DNA complexes with some ligands

In the range of millimeter wavelengths the dielectric properties of aqueous solutions of some biologically active ligands (potential anticarcinogen chlorophyllin; pharmacological drug caffeine; polyamine putrescine; mutagens proflavine and ethidium bromide; actinocin derivative, an analogue of antitumor antibiotic actinomycin D) and DNA complexes with these substances were studied. It was shown that complex formation is accompanied by the change in dielectric properties of the solution. These changes during interaction of DNA with the first three compounds correspond to a decrease in hydration (compared with the total hydration of free components), and in other cases they cause an increase in hydration. The number of water molecules bound with both the ligand and DNA nucleotide in the complex was estimated. The results were compared with existing models of DNA interaction with the studied substances.     

Ethidium bromide resistance in a rat liver epithelial cell line: Association with enhanced drug efflux

Ethidium bromide-resistant cell strains were obtained by continuous selection of an adult rat liver-derived cell line (ARL6T) grown in the continuous presence of 200 ngl ml ethidium bromide. Comparison of resistant strains and parental (sensitive) cells was made for uptake and binding of ethidium bromide, visualized as fluorescent ethidium bromide-nucleic acid complexes. Although uptake of ethidium bromide was similar in parental and resistant cells, efflux kinetics were markedly different. Over a three-hour period, parental (sensitive) cells maintained fluorescence following a short ethidium bromide pulse (100 g/ ml ethidium bromide). In contrast, ethidium bromide-resistant cell lines eliminated photographically detectable fluorescent complexes within three hours following pulse exposure to ethidium bromide. The rapid elimination of ethidium bromide fluorescent complexes in all (5) resistant cell strains examined supports an efflux mechanism as contributing to the resistance of ethidium bromide cytotoxicity in these cells.Abbreviations EtBr ethidium bromide - HBSS Hanks' balanced salt solution     

The action of structural analogues of ethidium bromide on the mitochondrial genome of yeast

We have studied the effects on the yeast mitochondrial genome of four analogues of ethidium bromide, in which the phenyl moiety has been replaced by linear alkyl chains of lengths varying from seven to fifteen carbon atoms. These analogues are more efficient than ethidium bromide in inducing petite mutants inSaccharomyces cerevisiae. The drugs also cause a loss of mtDNA from the cellsin vivo; however these analogues are in fact less effective inhibitors of mitochondrial DNA replicationper se, as shown by directin vitro studies. It is concluded that these analogues are more efficient than ethidium bromide in causing the fragmentation of mitochondrial DNA inS. cerevisiae.