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PubMed1982

Comparison of two flow cytometric assays for cellular RNA--acridine orange and propidium iodide.

Two flow cytometric assays for cellular RNA, two-step acridine orange (TSAO) and propidium iodide (PI), were compared with each other and with ultraviolet (uv) spectrophotometry of RNA to determine their ability to quantitate cellular RNA and to differentiate between proliferating and quiescent (Q) cells. The model system used for these comparisons was cells from unfed cultures of two mouse mammary tumor lines designated 66 and 67. The growth kinetics of unfed 67 and 66 cells were characterized by a decrease in cellular RNa (a factor of 2) with the decrease occurring throughout the entire population of cells as they entered plateau phase. The time course of the RNA decrease as monitored by the PI assay closely corresponded to that observed by uv spectrophotometric measurements. The TSAO assay, however, agreed with the uv spectroscopy and PI assays on the extent of the RNA decrease but showed no decrease in RNA until 24 hours after the other assays indicated that a significant decrease had occurred. The plateau cells from 67 and 66 unfed cultures had a greatly lowered RNA content and were predominately (greater than 97%) in the G1 phase of the cell cycle in terms of DNA content. However, when compared with exponentially growing cells, they were a distinct Q population. This distinction could be observed using either the TSAO or the PI assay. The TSAO assay provided better resolution of the Q population and has the added advantage of giving DNA distribution simultaneously. Therefore, both flow cytometric assays are particularly useful in this system; the TSAO for monitoring the Q population and the PI for determination of kinetic changes in total cellular RNA.

Acridine Orange↗
PubMed1985

Denaturation and condensation of DNA in situ induced by acridine orange in relation to chromatin changes during growth and differentiation of Friend erythroleukemia cells.

DNA in situ is progressively denatured when the cells or nuclei are treated with increasing concentration of acridine orange (AO). This transition can be monitored by flow cytometry as a decrease in green fluorescence. The complexes of denatured DNA and AO undergo immediate condensation and aggregation; this step is manifested by appearance of red luminescence and formation of precipitates that can be detected by electron microscopy. The precipitates form preferentially in heterochromatin as well as in ribosomes and polysomes. Their formation and further aggregation affects cellular light scatter properties in both the forward and right-angle direction. The AO-induced DNA denaturation and condensation was studied in nuclei of Friend erythroleukemia cells from exponentially growing, differentiated or quiescent cells. The DNA in nuclei of quiescent cells, from plateau-phase cultures, was the most sensitive to denaturation; it denatured (measured by changes in luminescence) at an AO concentration between 50 and 80 microM with the midpoint of the transition (Cd) at 70 microM. DNA in nuclei of differentiated cells (dimethyl-sulfoxide-induced erythroid differentiation) was more resistant (Cd = 77-83 microM), whereas DNA in exponentially growing cells was the most resistant (Cd = 86 microM). Extraction of proteins with 0.1 M HCl at 0 degree C abolished the differences between the cells and shifted the transition to a lower AO concentration (Cd = 46 microM). For comparison, the midpoint transitions representing condensation of free, nucleic acids measured as light scatter changes occurred at 13, 22, 31 and 53 microM of AO, for rRNA, tRNA, and denatured and native-calf thymus DNA, respectively. Denaturation and condensation of DNA, which can be induced by AO either in isolated nuclei or viable permeabilized or fixed cells provides a new approach to discriminate cell subpopulations with different chromatin structure by flow cytometry. The molecular mechanisms of this phenomenon are discussed.

Acridine Orange↗
PubMed1992

Two AgNOR counts in fine-needle aspirates of lymphoproliferative disorders compared with acridine orange flow cytometry.

Studies have shown that argyrophilic nucleolar organizer region-associated proteins (AgNORs) may correlate with DNA ploidy and/or proliferative activity in neoplastic and non-neoplastic conditions. However, studies have estimated only the mean AgNOR counts. Here we used two AgNOR counts, one of which may correlate with DNA ploidy and the other with proliferative activity. The mean AgNOR count (mAgNOR) was defined as the mean number of AgNORs/nucleus in 100 cells and may represent DNA or RNA index. The percentage of nuclei exhibiting 5 or more AgNORs/nucleus (pAgNOR) may reflect proliferative activity. These two AgNOR counts were correlated with results from acridine orange flow cytometry in 50 fine-needle aspirate (FNA) smears of nodal and extranodal sites, including three cases of reactive lymphadenopathy and 47 cases of non-Hodgkin's lymphoma. The mean mAgNOR count in the diploid specimens was 2.03 (+/- 0.74 SD) and 2.62 (+/- 0.73 SD) in the aneuploid tumors (P less than 0.0001). Samples with a low RNA index had mean mAgNOR of 1.80 (+/- 0.41 SD), whereas those with high RNA had a mean mAgNOR of 2.93 (+/- 0.86 SD) (P less than 0.0001). Lesions with low proliferative index, determined by flow cytometry, had a mean pAgNOR of 4%, whereas those with intermediate and high proliferative indices had a mean pAgNOR of 16% (P less than 0.0001). A similar but less significant correlation existed between RI and pAgNOR (P less than 0.005). We conclude that the two AgNOR counting methods may reliably reflect cell kinetics and distinguish ploidy from proliferative activity, making them useful adjuncts to flow cytometry in limited cytology specimens and small biopsy samples.

Acridine Orange↗
PubMed1978

Effects of an acridine half-mustard (ICR 191) on growth and ploidy of frog cells in culture.

The effects of an acridine half-mustard, ICR 191, on the growth rate and ploidy of four haploid and two diploid lines of Rana pipiens cells in culture were studied. Growth curves indicate that the haploid and diploid cell lines were equally resistant to a 4-hour exposure of this drug (0.1 micrometer to 10 micrometer. ICR 191 treatment induced the haploid cell cultures to become diploid. The proportion of diploid cells increased progressively with respect to time after the 4-hour exposure period. The greater the concentration of ICR 191 applied, the more rapid the rate of conversion. Autoradiographic determinations of percent labelled nuclei indicate that DNA synthesis was not inhibited in haploid or in diploid cells. Therefore, the increased proportion of diploid cells did not originate from the small percentage of diploid cells in the initial population. Instead the haploid cells were converted to diploid cells. Time lapse cinematography indicated that the conversion mechanism was other than cell fusion. Conversion to higher ploidy did not occur when diploid cell cultures were exposed to ICR 191.

Acridines↗
PubMed1978

Electronic structure--activity relationships of antibacterial acridines.

The antibacterial activity of a series of amino- and fluorinated acridines was studied in the framework of their electronic structures. To calculate the electronic structure, a simple Hückel molecular orbital theory was used. A statistical regression analysis revealed linear correlations between the activity and the electronic indexes, particularly the electron density at the ring nitrogen.

Acridines↗
PubMed1988

Freezing and thawing alter chromatin stability of ejaculated human spermatozoa: fluorescence acridine orange staining and Feulgen-DNA cytophotometric studies.

Cryopreservation and freezing-thawing effects on the fertilizing ability of human spermatozoa commonly are evaluated by post-thaw motility. Various studies have depicted the ultrastructural changes caused by freezing-thawing, yet the chromatin alterations have been studied very limitedly. Our aim was to determine the extent to which freezing-thawing alters the chromatin of human spermatozoa, using two analytical methods: acridine orange staining and Feulgen-DNA cytophotometric studies. Both methods revealed a dramatic effect of freezing-thawing on sperm chromatin: the native DNA content decreased as did the Feulgen-DNA content, and sperm surface area was reduced. These results indicate an effect on DNA, diminished accessibility for Feulgen, and a decrease in nuclear surface area and prompt us to hypothesize a relationship between an "overcondensation" state for sperm chromatin after freezing-thawing and a lower conception rate for human semen after cryostorage.

Acridine Orange↗
PubMed1981

Lymphocyte stimulation by concanavalin A studied by the fluorescent probe acridine orange.

Viable mouse thymocytes or spleen leucocytes stained with acridine orange (AO) were divided into one part used for stimulation, and the other part for control. Analysis of cellular green-fluorescence emission enabled physicochemical changes in lymphocytes to be detected after 30 min stimulation with the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM). No change in fluorescence was observed with the nonmitogenic reagent wheat germ lectin (WGL) or with allogeneic cell stimulation (MLR). When green fluorescence intensity of individual cells was monitored by microfluorimetry, 30 min stimulation with Con A induced an increase, whereas PWM induced a decrease. When analysed by fluorescence spectrophotometry, Con A induced a 2 nm blue shift in emission maximum and a decrease in polarization values.

Acridine Orange↗
PubMed1985

The use of acridine orange staining and image analysis to detect bacteriuria.

Fifty urine samples from a variety of urological patients were analysed using the fluorochrome acridine orange in an automated system. The results were compared with standard colony counts. The method has a potential value in the detection of significant infection particularly in population studies.

Acridine Orange↗
PubMed1991

Acridine Orange, a precipitant for sulfated glycosaminoglycans, causes mucopolysaccharidosis in cultured fibroblasts.

The purpose of the present investigation was to examine whether or not a di-cationic amphiphilic compound that is known (1) to be accumulated in lysosomes and (2) to form insoluble complexes with sulfated glycosaminoglycans (sGAG) in vitro, is able to interfere with the lysosomal degradation of sGAG, thus causing mucopolysaccharidosis (MPS) in cultured cells. Acridine Orange (AO) was chosen for this study since it is known to meet the above requirements. Cultured fibroblasts from rat cornea were exposed to AO (0.7 microM to 30 microM) for 72 h; tilorone served as reference compound. AO (1.75 microM to 10 microM) caused MPS in a concentration-dependent manner, higher concentrations were cytotoxic. MPS was demonstrated by cytochemical staining with cuprolinic blue and by measuring the intracellular accumulation of [35S]-GAG. The sGAG-complexing properties of AO were demonstrated by using it as a fixative for the intralysosomal sGAG accumulated in tilorone-treated cells. The present findings give support to the working hypothesis that the MPS induced by di-cationic amphiphilic drugs is due to the formation of insoluble sGAG-drug complexes, with the result that the sGAG become resistant to lysosomal degradation.

Acridine Orange↗
PubMed1978

Quinacrine and acridine-R banding without a fluorescence microscope.

A technique is described in which a standard fluorescence microscope equipped with a high-pressure mercury lamp is replaced with an ordinary laboratory microscope fitted with a quartz-iodine lamp. A dark field condenser and a set of three filters, including an FITC interference filter, complete a 'fluorescence' microscope. The microscope has proved itself satisfactory in the study of Y-chromatin, chromosome Q-bands including Q-polymorphism, and acridine-R band. It is very easy to operate and does not emit ultraviolet light, which might harm operators. Total cost of the quartz-iodine lamp's outfit, filters, and a dark-field condenser is much less than that of a standard fluorescence microscope. The cost is especially low when a laboratory microscope with a quartz-iodine lamp is already at hand. Spectrofluorometric studies of QM and Q indicate that the present system will show even better performance if an interference filter with a transmission range of about 400 to 440--450 nm is designed and used in combination with a 455--475 nm barrier filter.

Acridines↗
PubMed1977

Differential staining of Chironomus thummi giant chromosomes by treatment with acridine orange after mild acid hydrolysis.

The fluorescence of Chironomus thummi giant chromosomes stained by acridine orange after mild acid hydrolysis (1N HCl at 37 degrees) has been studied. After very short hydrolysis as well as in control preparations (untreated by HCl) all chromosome regions show green fluorescence. After long hydrolysis (10 min) all chromosome bands fluoresce red. Intermediate time of hydrolysis (2 min) give in all transcriptionally inactive bands including centromeric ones red fluorescence whereas transcriptionally active puffing regions are green. The possible mechanisms of differential staining of individual chromosome regions are discussed. It is suggested that transcriptionally active chromosome regions are less susceptible to the action of acid or that the difference in chromatin stainability after acid-AO treatment is due to the difference in chromatin packing. The applicability of this method to the study of functional states of chromatin, at least in giant chromosomes, is discussed.

Acridines↗
PubMed1975

[Discontinued treatment with BudR and staining with acridine orange: observation of R- or Q- or intermediary banding (author's transl)].

Discontinued treatments with BudR at different periods of the cellular cycle produce various chromosome banding after staining with acridine orange. In particular, it is possible to observe R- or Q- or an intermediary banding, simply by varying the time of incorporation of BudR. This implies that the amount of AT or GC bases present locally in DNA is not directly responsible for the banding observed. Furthermore it appears that a precise correlation exists between replication and R- or Q-banding: the DNA located at each group of bands replicates either early (R-bands) or late (Q-bands). But these timings overlap towards the middle of phase S: if the treatment is given at that time, it is possible to observe aspects intermediary between Q and R.

Acridines↗
PubMed1993

32P-postlabeling analysis of dibenz[a,j]acridine DNA adducts in mice: preliminary determination of initial genotoxic metabolites and their effect on biomarker levels.

N-Heterocyclic aromatics (NHA) are widely occurring environmental pollutants formed during the pyrolysis of nitrogen-containing organic chemicals. NHA are found in significant amounts in tobacco condensates, synthetic fuels, gasoline engine exhaust, and effluents from the heating of coal. Dibenz[a,j]acridine (DBA) is an example of NHA. The potency of many carcinogenic compounds is related, at least in part, to the efficiency of their biological activation. We undertook studies to determine which initial metabolites of DBA lead to the formation of high levels of carcinogen-DNA adducts in vivo. DBA and its metabolites, trans-DBA-1,2-dihydrodiol (DBA-1,2-DHD), trans-DBA-3,4-dihydrodiol (DBA-3,4-DHD), and trans-DBA-5,6-dihydrodiol (DBA-5,6-DHD), were applied to the skin of mice. DNA was isolated using enzyme-solvent extraction method. DNA was 32P-postlabeled under conditions of limiting [32P]ATP. In skin, DBA produced two distinct adducts. The same two adducts were seen when DBA-3,4-DHD was applied. In addition the total adduct level elicited by DBA-3,4-DHD was higher than that of parent compound. Two adducts were seen when DBA-5,6DHD was applied, but these were very different from adducts seen with DBA. These results suggested that activation of DBA to DNA-binding compounds in skin includes initial formation of DBA-3,4-DHD. The data support development of biomarkers for the exposure and effect of this compound, and also suggest that specific metabolic susceptibility markers might be able to predict populations at increased risk.

Acridines↗
PubMed1986

Different instability of nuclear DNA at acid hydrolysis in cancerous and noncancerous cells as revealed by fluorescent staining with acridine orange.

Ehrlich cancer cells and inflammatory cells in mouse ascitic fluid were hydrolyzed and stained with acridine orange (AO). The AO hydrolysis curves for G1/G2 + M phase cancer cells and inflammatory cells were differentially determined using flow cytometry by monitoring the metachromatic red-shifted fluorescence of the fluorochrome bound to the single-stranded DNA produced by acid hydrolysis. By computer fitting of the Bateman function to the hydrolysis curves, the kinetic parameters k1 (rate constant for the production of single-stranded DNA), k2 (rate constant for the degradation of the produced single-stranded DNA), and y0 (theoretical value of the single-stranded DNA present initially) were determined. It was found that the k2 value, which reflects the degree of DNA instability, was much higher for cancer cells in both the G1 and G2 + M phases than for inflammatory cells. This finding led us to develop a method for the differential AO staining of cancer cells and non-cancerous cells utilizing the different degree of DNA instability at acid hydrolysis. AO staining after hydrolysis with 2N HCl at 30 degrees C for 8.5 min was found to be the optimal method. In the 60 cases of human malignant epithelial and nonepithelial tumors tested, all of the malignant tumor cells emitted metachromatic red fluorescence, while all of the nonmalignant tumor cells (5 cases of benign tumor) and normal cells emitted orthochromatic green fluorescence when observed with a violet excitation light under a fluorescence microscope. This new technique can be a useful tool for the screening of malignancy in exfoliative cytology and also for basic cancer research.

Acridine Orange↗
PubMed1983

[Hydrophobic acridine dyes for fluorescence staining of mitochondria in living cells. 1. Thermodynamic and spectroscopic properties of 10-n-alkylacridine orange chlorides].

10-n-Alkyl-acridine-orange-chlorides (alkyl-AOs) are excellent dyes for fluorescence staining of mitochondria in living cells. The thermodynamic and spectroscopic properties of the series alkyl = methyl to nonyl have been investigated. The dyes form dimers in aqueous solution. The dimerisation is mainly a consequence of the hydrophobic interaction. The dissociation constant K respectively association constant K-1 of the dimers describes the hydrophobic interaction and therefore the hydrophobic properties of the dye cations. The dissociation constant K = K0 at the standard temperature T = 298 K has been determined spectroscopically in aqueous solution. It depends on the length of the alkyl residue n-CmH2m + 1 (m = 1 - 9) (Table 2). In addition the standard dissociation enthalpies (energies) delta H0 and dissociation entropies delta S0 have been determined from the temperature dependence of K (Table 2). With increasing chain length m the thermodynamic parameters K0, delta H0, delta S0 decrease. Therefore with growing m the dimers are stabilized. This stabilization is an entropic effect which is diminished by the energetic effect. The change of the thermodynamic parameters with m is in agreement with the concept of hydrophobic interaction and the stabilization of water structure in the surroundings of hydrophobic residues. As one would expect nonyl-AO is the most hydrophobic dye of the series. As an example the spectroscopic properties of nonyl-AO have been determined. We measured the absorption, luminescence and polarization spectra in rigid ethanol at 77 K. Under these conditions alkyl-AOs associate like dyes in Water at room temperature. The spectra depend on the concentration of the solution. In very dilute solution we observe mainly the spectra of the monomers M, in concentrated solution the spectra of the dimers D. The spectra of M and D are characteristically different. The monomers have one long wave length absorption M1 = 20.000 cm-1 with resonance fluorescence. In addition there is a long living phosphorescence at 16.600 cm-1. Its polarization is nearly perpendicular to the plane of the AO residue. The dimers have two long wave length absorption bands D1 = 18.700 and D2 = 21.200 cm-1 with very different intensities. D1 has very low intensity and is forbitten, D2 is allowed. D1 shows fluorescence. Phosphorescence has not been observed. D1, D2 and also M1 are polarized in the plane of the AO residue. At short wave length absorption and polarization spectra are very similar. From the spectra we constructed the energy level diagram of M and D (Fig. 9). The first excited state of M splits in D in two levels. The level splitting and the transition i

Acridine Orange↗
PubMed1977

Distribution of acridine orange-stained RNA in neuroblastoma cells during differentiation.

Vital staining of neuroblastoma cells with acridine orange produces a bright intracellular red-orange fluorescence most probably due to the occurrence of RNA. The distribution of this fluorescence depends on the state of morphological differentiation. The fluorescence is predominantly found in the perikaryon, the growth cones, and the endings of the processes of differentiated cells. This is of special interest in respect to the biochemistry of differentiation and the function of nerve cells. Comparative autoradiographical studies with 3H-uridine demonstrate that the newly synthesised RNA is transported into the endings of the cell processes.

Acridines↗