Targeting of mitochondria by 10-N-alkyl acridine orange analogues: role of alkyl chain length in determining cellular uptake and localization

10-N-Nonyl acridine orange (NAO) is used as a mitochondrial probe because of its high affinity for cardiolipin (CL). Targeting of NAO may also depend on mitochondrial membrane potential. As the nonyl group has been considered essential for targeting, a systematic study of alkyl chain length was undertaken; three analogues (10-methyl-, 10-hexyl-, and 10-hexadecyl-acridine orange) were synthesized and their properties studied in phospholipid monolayers and breast cancer cells. The shortest and longest alkyl chains reduced targeting, whereas the hexyl group was superior to the nonyl group, allowing very clear and specific targeting to mitochondria at concentrations of 20-100 nM, where no evidence of toxicity was apparent. Additional studies in wild-type and cardiolipin-deficient yeast cells suggested that cellular binding was not absolutely dependent upon cardiolipin.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange.

Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and muscopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracelluar dye contents as low as 5 X 10(-16) mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

10N-nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria.

The acridine orange derivative, 10N-nonyl acridine orange, is an appropriate marker of the inner mitochondrial membrane in whole cells. We use membrane model systems to demonstrate that 10N-nonyl acridine orange binds to negatively charged phospholipids (cardiolipin, phosphatidylinositol and phosphatidylserine). The stoichiometry has been found to be 2 mol 10N-nonyl acridine orange/mol cardiolipin and 1 mol dye/mol phosphatidylserine or phosphatidylinositol, while, with zwitterionic phospholipids, significant binding could not be detected. The affinity constants were 2 x 10(6) M-1 for cardiolipin-10N-nonyl-acridine-orange association and only 7 x 10(4) M-1 for that of phosphatidylserine and phosphatidylinositol association. The high affinity of the dye for cardiolipin may be explained by two essential interactions; firstly an electrostatic interaction between the quaternary ammonium of nonyl acridine orange and the ionized phosphate residues of cardiolipin and secondly, hydrophobic interactions between adjacent chromophores. A linear relationship was demonstrated between the cardiolipin content of model membranes and the incorporated dye. Consequently, a convenient and rapid method for cardiolipin quantification in membranes was established and applied to the cardiolipin-containing organelle, the mitochondrion.     

Discrimination of respiratory dysfunction in yeast mutants by confocal microscopy, image, and flow cytometry

Living yeast cells can be selectively stained with the lipophilic cationic cyanine dye DiOC6(3) in a mitochondrial membrane potential-dependent manner. Our study extends the use of flow cytometric analysis and sorting to DiOC6(3)-stained yeast cells. Experimental conditions were developed that prevented the toxic side effect of the probe and gave a quantitative correlation between fluorescence and mitochondrial membrane potential, without any staining of other membranes. The localization of the fluorochrome was checked by confocal microscopy and image cytometry. The mitochondrial membrane alterations were also tested through cardiolipin staining with nonyl acridine orange. Differences in light scattering and in fluorescence were detected in mutants (rho-, rho degrees, mit-, or pet-) and wild-type (rho+mit+) populations of yeast. The dye uptake of respiratory-deficient yeast strains was significantly reduced as compared to that of the wild-type. Application of an uncoupler (mCICCP), which collapsed the mitochondrial membrane potential (alphapsi(m)), led to a drastic reduction of the dye uptake. It was observed that a decrease in deltapsi(m), was usually correlated with a decrease in cardiolipin stainability by nonyl acridine orange (NAO). Quantitative flow cytometry is a fast and reproducible technique for rapid screening of yeast strains that might be suspected of respiratory dysfunction and/or mitochondrial structural changes. We give evidence that it is an adequate method to characterize and isolate respiratory mutants through sorting procedure, with selective enrichment of the population studied in respiring or non-respiring yeast cells. Confocal microscopy and image cytometry corroborate the flow cytometry results.     

Bertalanffy-like fluorescence staining with 3-dimethylamino-6-methoxyacridine

Three new acridine dyes, 3-dimethylamino-6-methoxyacridine 1, 3-amino-6-methoxyacridine 2 and 3-amino-7-methoxyacridine 3, have been prepared and tested as fluorochromes of LM- and HeLa-cells. The dyes are basic compounds (pKA: 1 8,76; 2 8,01; 3 7,65) and form cations in neutral or acidic aqueous solutions by addition of a proton to the aza-nitrogen atom of the heterocycle. The fluorochromes stain fixed LM- and HeLa-cells at pH = 6. The fluorescence shows metachromasy similar to the staining with acridine orange AO according to the technique of Bertalanffy. But there is less fading of the fluorescence. The dye 1 is the most suitable fluorochrome of the series. It was studied in detail. Using optimized staining conditions the fluorescence of the nucleus is yellow-green that of the cytoplasm and the nucleoli orange or brownish-red. Enzymatic digestion experiments show that the dye cations are bound to DNA in the nucleus and to RNA in the cytoplasm or nucleoli. The absorption and emission spectra of the stained cells have been studied by means of microspectrophotometry. The absorption spectra of the nucleus and the cytoplasm are very similar. The maximum of the long wave length absorption of both occurs at 21400 cm-1 (467 nm) with a shoulder at ca 20100 cm-1 (498 nm). The fluorescence spectra of nucleus and cytoplasm of metachromatically stained cells are different. The emission maximum of the cytoplasm and nucleoli, 16200 cm-1 (617 nm), is red-shifted relative to the maximum of the nucleus, 18200 cm-1 (549 nm). This shift causes the metachromatic fluorescence effect. In addition we studied the concentration dependence of the absorption and fluorescence spectra of the cation 1 in aqueous solution, pH = 6, in the concentration range 6 X 10(-6)-6 X 10(-4) M. Shape and maximum of the long wave length absorption and emission depend only slightly on the concentration: Mean value of absorption maximum ca 21500 cm-1 (465 nm), shoulder at ca 20300 cm-1 (493 nm), fluorescence maximum ca 18300 cm-1 (547 nm). With growing concentration diminishes the molar absorptivity. This decrease in absorptivity and isosbestic points in the absorption spectra indicate the formation of dimers with growing dye concentration. The absorption spectra of the metachromatically stained cells and of the dye in aqueous solution are very similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of neoplastic cells in the bloodstream]

A study was made of the efficacy of trypan blue, acridine orange, tetracycline and oxytetracycline for detection of tumour cells injected into the blood stream of rats. The cells were identified in the mesenteric microvessels by intravital microscopy. Fluorescence of fluorochromized cells was observed in the blue-violet (lambda max = 400 nm) and ultra-violet (lambda max = 365 nm) irradiation of the fluorescent lamp and in the laser irradiation (lambda = 337 nm). The cells stained with acridine orange had a higher fluorescence intensity and a more distinct structure than those labelled with tetracyclines. Identification of cells with trypan blue was more difficult. The fluorescent method of determination is rather simple and permits to indentify tumour cells directly in the blood stream.     

Use of nonyl acridine orange and rhodamine 123 to follow biosynthesis and functional assembly of mitochondrial membrane during L1210 cell cycle.

Specific mitochondrial incorporation of 10 N-nonyl acridine orange (NAO) is demonstrated by subcellular fractionation of rat hepatocytes. Moreover, comparative studies with NAO and rhodamine 123 (Rh 123) prove that acridine orange-derivative uptake is independent of transmembrane mitochondrial potential, a property allowing its utilization for the assessment of mitochondrial membrane mass modifications under various physiological states. Using NAO and Rh 123, we have respectively followed the biosynthesis of mitochondrial membrane and its assembly under a functional state during the L1210 cell cycle. Their evolution occurs in two stages according to a well-defined sequential order. Mitochondrial biogenesis, as revealed by NAO incorporation, occurs essentially in the G1 phase (probably mitochondrion enlargement) but also starts in late S phase (probably mitochondrion division). The increased amount of functional mitochondrial membrane, monitored by Rh 123 uptake, is emphasized in late G1 (prerequisite to DNA synthesis) and during G2M phases (prerequisite to mitosis). This alternative succession of phases displays the existence of a time-lag between the biosynthesis of mitochondrial membrane and its functional organization. Such an analysis confirms the potential of the NAO probe to evaluate mitochondrial membrane mass changes in various biological fields.     

Nature and distribution of constitutive heterochromatin and NOR location in the grasshopper Phaeoparia megacephala (Romaleidae: Orthoptera)

The constitutive heterochromatin (CH) of Phaeoparia megacephala was studied using C-banding and fluorochrome staining (CMA3, DAPI and acridine orange). The nucleolar organizer regions (NOR) were identified with silver staining. The chromosome complement of this species was 2n = 23, XO in males, and 2n = 24, XX in females. The CH was pericentromeric in all chromosomes. L1, L2, L3 and X chromosomes showed large blocks of CH, while the medium and small chromosomes had small blocks. The staining procedure with acridine orange revealed the same pattern. All the pericentromeric regions showed small blocks of CMA3-positive constitutive heterochromatin (GC-rich regions), while only part of the large C-band positive chromosome segments (L1, L2, L3 and X) were CMA3 positive. This character demonstrates an uncommon heterogeneity of constitutive heterochromatin in P. megacephala. The fluorochrome DAPI did not reveal DAPI-positive regions (AT-rich regions). Silver staining revealed only one pair of medium chromosomes with NOR.     

Bid-cardiolipin interaction at mitochondrial contact site contributes to mitochondrial cristae reorganization and cytochrome C release

Release of cytochrome c from the mitochondrial intermembrane space is critical to apoptosis induced by a variety of death stimuli. Bid is a BH3-only prodeath Bcl-2 family protein that can potently activate this efflux. In the current study, we investigated the mitochondrial localization of Bid and its interactions with mitochondrial phospholipids, focusing on their relationships with Bid-induced cytochrome c release. We found that Bid binding to the mitochondria required only three of its eight helical structures (alpha4-alpha6), but not the BH3 domain, and the binding could not be inhibited by the antideath molecule Bcl-x(L). Membrane fractionations indicated that tBid bound to mitochondrial outer membranes at both contact and noncontact sites. Bid could interact with specific cardiolipin species on intact mitochondria as identified by mass spectrometry. Like the binding to the mitochondria, this interaction could not be blocked by the mutation in the BH3 domain or by Bcl-x(L.) However, a cardiolipin-specific dye, 10-N-nonyl acridine orange, could preferentially suppress Bid binding to the mitochondrial contact site and inhibit Bid-induced mitochondrial cristae reorganization and cytochrome c release. These findings thus suggest that interactions of Bid with mitochondrial cardiolipin at the contact site can contribute significantly to its functions.     

Lysosome-like alpha-granules of thrombocytes: detection by acridine orange treatment, properties and transformations during the course of blood coagulation reactions]

Using the ability of lysosome-like alpha-granules of thrombocytes to accumulate flurochrome acridine orange, their transformation kinetics was studied in the course of cell viscous metamorphosis utilizing the fluorometric technique. It turned out that during the thrombin induced cellular viscous metamorphosis lysosomes released their contents: the process is stimulated by the presence of extracellular Ca2+. Competitive relations were revealed between the accumulation within cytoplasmic granules of thrombocytes of acridine orange and serotonin, and that of histamine and novocaine. The process of fluorochrome accumulation was found to be temperature dependent and to be inhibited by monoiodacetate.     

Thermodynamics of mucopolysaccharide–dye binding. I. Identification of free and bound dye via membrane filtration: Acridine orange–dermatan sulfate system

Stoichiometric mixtures of acridine orange with dermatan sulfate at total dye concentrations ? 1 × 10^?5 M show fluorescence maxima at 540 nm and 660 nm on excitation at 436 nm. By means of membrane filtration, it is directly demonstrated that the species emitting at 540 nm is due only to unbound dye whereas the 660-nm emitting species is due to bound dye. It is, therefore, possible to differentiate unbound acridine orange from its dermatan sulfate complex solely by spectroscopic methods. Thermodynamic binding parameters can be calculated from rapid spectroscopic measurements without disturbing the system.     

Titration of cardiolipin by either 10-<Emphasis Type="Italic">N</Emphasis>-nonyl acridine orange or acridine orange sensitizes the adenine nucleotide carrier to permeability transition

Under the action of carboxyatractyloside or fatty acids, adenine nucleotide translocase switches its function from nucleotide carrier to modulator of the opening of a non-specific pore. In addition to the effect of these agents, this modification in activity is, in some way, dependent on the influence of the lipid milieu of the membrane. Cardiolipin is, among other membrane phospholipids, the one that interacts the most with the translocase. This work shows that 10-N-nonyl acridine orange and acridine orange, probes for this phospholipid, modify the sensitivity of the translocase to carboxyatractyloside, oleate, and palmitate to induce permeability transition. The results also show that these probes stimulate the release of mitochondrial cytochrome c, and increase labeling of the carrier by eosin 5-maleimide. Based on the aforementioned it is proposed that the increase in sensitivity is due to a conformational change in the translocase, induced by the binding of the probe to cardiolipin.     

Fluorochrome staining of multilamellar liposomes.

Multilamellar liposomes can be stained with such fluorochromes as acridine orange, eosin Y, neutral red, and thiazine red. The liposomes are brought into a 1% solution of the fluorochrome; 5-10 minutes later they are centrifuged and washed by resuspending in water or phosphate buffered saline three times. The last pellet is resuspended and a drop studied with the fluorescence microscope (1000 x magnification). The fluorochrome is seen to be accumulated in the liposomal membranes. Acridine orange could also be trapped in the aqueous compartments of the liposomes but the trapped fluorochrome was gradually lost from the liposomes. Part of the fluorochrome, however, remained associated with the liposomal membranes for a long time. Additional experiments justify the conclusion that an equilibrium is maintained between fluorochromes in the aqueous and lipid phases.     

Absorption and Fluorescence Spectra of Euchrysine Ggnx and Acridine Orange; Effects of Heparin, NaCl and a Cation Exchange Resin

The absorption and fluorescence spectra of two samples of dye labeled euchrysine were found to differ. One sample, labeled GGNX, had absorption and fluorescence maxima of 435 and 515 nanometers (nm) respectively. The other sample was not further labeled, but had absorption and fluorescence maxima of 492 and 535 nm. The latter values, as well as the shape of both the fluorescence and absorption curves of the second sample were superimposable on a recrystallized sample of acridine orange labeled correctly C. I. 46905. Euchrysine has two free amino groups which are fully methylated in acridine orange, therefore a nitrous acid test can differentiate the two dyes. The sample of euchrysine labeled GGNX gave a reaction, as did acridine yellow, C. I. 46025, but acridine orange, C. I. 46005, did not. Fluorescence metachromasy of euchrysine is less efficient than that of acridine orange in two ways: the shift in the spectrum is smaller by about 40 nm, making the separation of the colors more difficult both visually and by instruments and the metachromatic fluorescence has less than half of the intensity of acridine orange as measured at the peak for each dye. Confusion between these two dyes has occurred because suppliers have used the names interchangeably. For critical studies, the dye used should be identified by its Colour Index number.     

Fluorochrome Staining of Multilamellar Liposomes

Multilamellar liposomes can be stained with such fluorochromes as acridine orange, eosin Y, neutral red, and thiazine red. The liposomes are brought into a 1% solution of the fluorochrome; 5-10 minutes later they are centrifuged and washed by resuspending in water or phosphate buffered saline three times. The last pellet is resuspended and a drop studied with the fluorescence microscope (1000 × magnification). The fluorochrome is seen to be accumulated in the liposomal membranes.

Acridine orange could also be trapped in the aqueous compartments of the liposomes but the trapped fluorochrome was gradually lost from the liposomes. Part of the fluorochrome, however, remained associated with the liposomal membranes for a long time.

Additional experiments justify the conclusion that an equilibrium is maintained between fluorochromes in the aqueous and lipid phases.     

Luminescence parameters of nuclear blood cells during the immune response process

The synthetic activity of nuclear blood cells in the process of immunization of rabbit with ovalbumin was studied by luminescence microspectroscopy . We followed the parameter alpha, the ratio between the intensities of the red (640 nm) and green (530 nm) components of the luminescence spectrum of acridine orange fluorochrome cells. The antiserum titre was determined too. The data obtained make it possible to correlate the increase in alpha of the immunocompetent cells with the content of antibodies synthesized by these cells in response to the protein antigen. It has been shown that within definite limits the parameter alpha does determine the synthetic activity of the immunocompetent cells.     

Cytogenetic Studies on Workers of the Neotropical Ant Wasmannia auropunctata (Roger 1863) (Hymenoptera: Formicidae: Myrmicinae)

Wasmannia auropunctata is known as one of the worst invasive ants in the World. A cytogenetic study was conducted on two native populations from southeastern Bahia, Brazil. The analysis of the chromosomes observed in mitotic metaphases was made by a combination of methods: Giemsa conventional staining, chromomycin A3 (CMA3) and 4-6-diamidino-2-phenylindole (DAPI) fluorochrome staining, and acridine orange banding. The workers have all the karyotype 2n=32, with ten pairs of metacentric and six pairs of acrocentric chromosomes. One chromosome arm of the pair ten was positive for CMA3 and acridine orange, suggesting the occurrence of a nucleolar organizing region. This region is an interesting marker because is very conservative and seems to constitute an interesting specific taxonomic character. The pericentromeric region of many chromosomes was stained with DAPI, evidencing the occurrence of AT bases rich heterochromatin.     

10-N nonyl-acridine orange: a fluorescent probe which stains mitochondria independently of their energetic state

The specificity of binding of 10-N Nonyl Acridine Orange to mitochondria, and more precisely to inner membranes, is demonstrated by subcellular fractionation of hepatocytes. Unlike Rhodamine 123, which is a preferential marker of the transmembrane potential, Nonyl Acridine Orange binding is essentially independent of the mitochondria energization state although a low uptake of this dye, in response to the potential, may be measured. So 10-N Nonyl acridine orange is an appropriate marker of the mitochondial membrane surface per unit of cell mass.     

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.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange

Summary Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and mucopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracellular dye contents as low as 5×10^–16 mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.