Induction of cytoplasmically inherited respiration-deficient ('petite') mutants by photodynamic action of acridine compounds

All acridines used (acriflavine, proflavine, acridine orange and 3-azido-10-methylacridinium chloride) produced killing in yeast cells when activated with visible light. Acriflavine, proflavine and 3-azido-10-methylacridinium chloride, but not acridine orange, produced petite and sectored colonies. Both cell killing and petite induction by light activation of acriflavine resulted apparently from photodynamic action mediated by singlet oxygen (1O2) since the effect were prevented by either sodium azide or anaerobiosis. The biological effects of 3-azido-10-methylacridinium chloride, which was developed as a potential photoaffinity probe for studying the binding and biological effects of acridines, appeared to be due to a photodynamic action analogous to that of acriflavine. Sodium azide or anaerobiosis prevented the light-activated effects of 3-azido-10-methylacridinium chloride despite the fact that the initial chemical breakdown of the azido derivative induced by light was not affected. Cells suspended in D2O demonstrated an enhanced response to 3-azido-10-methylacridinium chloride with irradiation. These results indicate that singlet oxygen mediates the light-activated biological effects of both acriflavine and 3-azido-10-methylacridinium chloride.     

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.     

Comparison of petite induction in yeast by acridines,ethidium and their photoaffinity probes

The production of petite mutations by different acridine analogs was studied in Saccharomyces cerevisiae. Compounds with amino substituents at the 2 and 3 positions of the acridine nucleus and methylation at position 10 were effective for petite induction in growing cells but not in resting cells, while those with chloro, nitro and methoxy substituents were not effective in either resting or growing cells.Photosensitive azido derivatives of the acridines were tested to evaluate the role of covalent drug attachment for mutagenesis in resting cells. Photolysis of resting cells with 9-azido, 3-azido-6-amino-, 9-azido-10-methyl-, or 3-azido- 6-amino-10-methyl-acridine was highly toxic. 3-Azido-6-amino-acridine, and especially 3-azido-10-methyl-, and 3-azido-6-amino-10-methyl-acridine, were effective petite inducers in resting cells. Thus, the photosensitive (azido) group at position 9 produced only cell killing while the azido group at position 3 and/or 6 led to effective petite induction in resting cells. In contrast, petite induction was observed only for growing cells, for dark control experiments with these compounds or with the monoazide precursor compounds.     

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.     

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.     

The influence of acridine dyes and caffeine on recovery from ultraviolet damage in Eudorina elegans.

Caffeine and the acridine dyes, acridine orange and acriflavine, were used to examine the repair potential in Eudorina elegans following ultraviolet irradiation. Acridines blocked photoreactivation primarily as a result of absorption of photoreactivating wavelengths, but acridines did not influence dark survival. Therefore, an acridine-sensitive excision-resynthesis-repair process is absent in Eudorina. Caffeine decreased both dark and light survival, the latter only after relatively high doses of ultraviolet light were used for inactivation. The caffeine-sensitive repair process appears to function most actively when the organisms are engaged in DNA synthesis, indicating that a postreplication-repair system exists in Eudorina. However, the data suggest that a repair system not associated with the DNA synthetic phases may also exist.     

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.     

Identification of the 9-aminoacridine/DNA complex responsible for photodynamic inactivation of P22

Acridine dyes bound to the condensed DNA within phage particles sensitize them to inactivation by visible light. The mechanism involves absorption of photons by an acridine/DNA complex, generating singlet oxygen, which covalently damages nearby proteins needed for DNA injection [Bryant, J., & King, J. (1985) J. Mol. Biol. 180, 837-863]. Acridines and related dyes interact with double-stranded DNA through a number of binding modes. To determine in condensed phage DNA the binding mode responsible for this inactivation, we have studied the formation of the DNA/acridine target complexes for photoinactivation. Analysis of the kinetics of 9-aminoacridine binding to Salmonella phage P22 particles revealed the formation of two binding species, one of which appeared more rapidly and was apparently an intermediate in the formation of the second. The rapidly forming species represented DNA sites with intercalated acridines, while the more slowly forming species represented the subsequent binding of additional acridine molecules to the DNA backbone of sites already containing intercalated dye. The rates of photoinactivation correlated with the rate of binding of 9-aminoacridine to the DNA backbone. This suggests that the most effective species for sensitizing phage to light-induced damage has acridine molecules stacked alongside the backbone of a region with intercalated molecules.     

Stimulation of incorporation of nucleic acid precursors into HeLa cells caused by provaline

1. The effect of proflavine and other acridines on the incorporation of precursors into the nucleic acids of HeLa cells was examined. 2. Relatively low concentrations (50mum) of proflavine completely inhibited incorporation of precursors into DNA, but allowed a small extent of incorporation into RNA. 3. Acridine-resistant incorporation into RNA was unaffected by actinomycin D at 2mug./ml. and persisted even at high concentrations (500mum) of many acridines. 4. A few combinations of acridine and precursor, notably 250mum-proflavine and [(14)C]adenine, caused a stimulation of incorporation. 5. The proflavine-stimulated incorporation was into alkali-stable di- and tri-nucleotides. 6. It was concluded that the effect was due to the preferential inhibition of degradation of a fraction of RNA that normally turned over, thus allowing small radioactive oligonucleotides to accumulate in the cells.     

Interactions of chlorophyllin with acridine orange,quinacrine mustard and doxorubicin analyzed by light absorption and fluorescence spectroscopy

The present study was designed to estimate the ability of chlorophyllin (CHL) to interact with two acridine mutagens, quinacrine mustard (QM) and acridine orange (AO), and with the antitumor anthracycline doxorubicin (Dox). To this end, aqueous solutions of QM, AO or Dox during titration with CHL were subjected to spectrophotometry and spectrofluorimetry to detect possible interactions between these reagents. The data indicate that CHL forms complexes with AO, QM or Dox in these solutions. The presence of the complexes was manifested by a bathochromic shift of the absorption spectra, as well as by strong quenching of the fluorescence of each of these mutagens in the presence of CHL. CHL, thus, may serve as an interceptor of these mutagenic acridines in different in vivo or in vitro applications. Its ability to interact with Dox may potentially be utilized to detoxify patients overdosed with this or similar drugs.     

The genetic toxicology of acridines

Acridine and its derivatives are planar polycyclic aromatic molecules which bind tightly but reversibly to DNA by intercalation, but do not usually covalently interact with it. Acridines have a broad spectrum of biological activities, and a number of derivatives are widely used as antibacterial, antiprotozoal and anticancer drugs. Simple acridines show activity as frameshift mutagens, especially in bacteriophage and bacterial assays, by virtue of their intercalative DNA-binding ability. Acridines bearing additional fused aromatic rings (benzacridines) show little activity as frameshift mutagens, but interact covalently with DNA following metabolic activation (forming predominantly base-pair substitution mutations). Compounds where the acridine acts as a carrier to target alkylating agents to DNA (e.g. the ICR compounds) cause predominantly frameshift as well as base-pair substitution mutations in both bacterial and mammalian cells. Nitroacridines may act as simple acridines or (following nitro group reduction) as alkylating agents, depending upon the position of the nitro group. Acridine-based topoisomerase II inhibitors, although frameshift mutagens in bacteria and bacteriophage systems, are primarily chromosomal mutagens in mammalian cells. These mutagenic activities are important, since the compounds have considerable potential as clinical antitumour drugs. Although evidence suggests that simple acridines are not animal or human carcinogens, a number of the derived compounds are highly active in this capacity.     

Effect of Deoxyribonucleic Acid Ligands on Deoxyribonucleases and Deoxyribonucleic Acid Polymerase I of Escherichia coli K-12

Endonuclease I, exonuclease I, and exonuclease II-deoxyribonucleic acid (DNA) polymerase I activities are not vital functions in Escherichia coli, although the latter two enzymes have been indirectly shown to be involved in DNA repair processes. Acridines such as acridine orange and proflavine interfere with repair in vivo, and we find that such compounds inhibit the in vitro activity of exonuclease I and DNA polymerase I but stimulate endonuclease I activity and hydrolysis of p-nitrophenyl thymidine-5′-phosphate by exonuclease II. Another acridine, 10-methylacridinium chloride, binds strongly to DNA but is relatively inert both in vivo and in vitro. These experiments suggest that acridines affect enzyme activity by interacting with the enzyme directly as well as with DNA. Resulting conformational changes in the DNA-dependent enzymes might explain why similar acridines which form similar DNA complexes have such a wide range of physiological effects. Differential sensitivity of exonuclease I and DNA polymerase I to acridine inhibition relative to other DNA-dependent enzymes may contribute to the acridine sensitivity of DNA repair.     

Comparative studies of the effects of acridines and other petite inducing drugs on the mitochondrial genome of Saccharomyces cerevisiae.

Summary The effects of the acridines euflavine and proflavine on mitochondrial DNA (mtDNA) replication and mutation inSaccharomyces cerevisiae have been compared. In contrast to previous results we found that under our conditions proflavine can indeed induce high levels (>80%) of petite mutants, although six times less efficiently than euflavine. The parameters measured for mutagenesis of the mitochondrial genome and inhibition of mtDNA replication in whole cells suggest that the modes of action of euflavine and proflavine are very similar. After extended (18h) treatment of growing cells with each drug the percentage loss of mtDNA or genetic loci was almost coincidental with the extent of petite induction.It was found that proflavine is equally as effective as euflavine in inhibiting mtDNA replication in isolated mitochondria in contrast to the differential between the drugs observed in vivo. However, proflavine and euflavine inhibit cellular growth at almost the same concentrations. It is therefore proposed that there is some intracellular permeability barrier which impedes proflavine access to the mitochondrial DNA replicating system.The petites induced by euflavine (and proflavine) are characterized by there being a preferential induction ofrho ⁰ petites lacking mtDNA as opposed torho ^- petites retaining mtDNA. This is in contrast to the relative proportions of such petites induced by ethidium bromide or berenil. A scheme for the production of petites by euflavine is presented, in which euflavine inhibits the replication of mtDNA, but does not cause direct fragmentation of mtDNA (unlike ethidium bromide and berenil). The proposed scheme explains the production of the high frequency ofrho ^o cells, as well as therho ^- cells induced by euflavine. The scheme also accounts for previous observations that euflavine only mutants growing cultures, and that the buds, but not mother cells, become petite.     

A comparison of the bactericidal and photobactericidal activities of aminoacridines and bis(aminoacridines)

An attempt was made to increase the activity of some antibacterial acridines in two ways: (a) via the use of bis-aminacrines (analogues of 1,6-bis-[9-acridinamino]hexane), in order to increase the potential for DNA intercalation and (b) via photoactivation, as several acridines are known to produce the cytotoxin singlet oxygen on irradiation. The bactericidal activity of the simple aminoacridines was much higher than that of the corresponding bis-acridine compounds. In addition, on low-power illumination of the compounds in liquid culture, many of the monomeric compounds exhibited marked increases in activity against both Gram-negative and Gram-positive bacteria, whereas the bis-acridines showed little or no increase.     

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.     

Inhibition of Pyrimidine Dimer Formation in DNA by Cationic Molecules: Role of Energy Transfer

In addition to the acridine dyes, acridine orange and proflavine, we find that three other cationic molecules which bind to DNA-ethidium bromide, chloroquine, and methyl green-inhibit the production of cyclobutyl pyrimidine dimers by ultraviolet radiation. Intercalation is not necessary for dimer inhibition. The long range nature of the inhibition implies that energy transfer is responsible. The transfer is between the lowest excited singlet state of DNA and the acceptor singlet, and seems to involve the F?rster mechanism.     

Terminations of DNA synthesis on 'proflavine and light'-treated phi X174 single-stranded DNA

Bacteriophage phi X174 single-stranded DNA molecules were primed with five different restriction fragments and irradiated with visible light in the presence of proflavine. This photodamaged DNA was used as template for the in vitro complementary chain synthesis by E. coli DNA polymerase I (Klenow fragment). Chain terminations were observed by polyacrylamide gel electrophoresis of the synthesized products and localized by comparison with standard sequencing performed simultaneously on the untreated template. 90% of the chain terminations occurred one nucleotide before a guanine residue in the template strand. More than 80% of the sequenced guanine residues were blocking lesions demonstrating the absence of 'hot-spots' for the photodamaging effect of proflavine. At a defined position, the chain termination frequency increased linearly with the irradiation time and was directly influenced by the proflavine concentration present. An important part of lesions resulted from the action of singlet oxygen produced by excited proflavine as shown by the effect that both NaN3 and 2H2O exerted on the reaction. The induced blocking lesions must be important in vivo since no complete replicative forms could be extracted from cell infected with bacteriophages inactivated by 'proflavine and light' treatment.     

On the complex formation of acridine dyes with DNA. VII. Dependence of the binding on the dye structure

The binding constants for the complex formation of more than twenty ring nitrogen-and amino-substituted acridine derivatives with calf thymas DNA were measured by a fluorescence method. DNA quenches the fluorescence of the aminoacridine dyes so long as both amino hydrogens are not substituted. These dyes show an enhancement of their fluorescence intensity in the presence of DNA. Typical representatives of both are proflavine and acridine orange derivatives, respectively. A discussion of steric and electronic influences of various substituents attached to the ring nitrogen and amino groups on the binding led to the concept of different conformations for intercalated acridines without amino groups and the aminoacridines. The electrostatic binding site of the former seems to be the positively charged ring nitrogen, while the binding sites in the aminoacridines are so located that the amino groups are directed towards the negatively charged DNA phosphates.     

Cytogenetic assays of chemical clastogens using mammalian cells in culture.

A protocol is described for cytogenetic assays of chemical mutagens using mammalian cells in vitro. The system employs continuous drug treatment (3 concentrations) for up to 8 h and recovery-cell populations after pulse treatments with a high dose. Both direct fixation (for recording spindle anomalies in anaphase) and colcemid-hypotonic fixation (for reading metaphase chromosome aberrations) are used in order to estimate the effects of an agent as a mitotic poison and as a clastogen respectively. Some DNA intercalating dyes (acridine orange, quinacrine mustard, neutral red) were found to be highly clastogenic whereas others (quinacrine dihydrochloride, 33258 Hoechst) are not.     

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.