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Cytostatic and cytotoxic properties of pyronin Y: relation to mitochondrial localization of the dye and its interaction with RNA.

Pyronin Y (PY) is an intercalating cationic dye that shows specificity towards RNA. In viable cells this dye also accumulates in mitochondria. The cytostatic and cytotoxic effects of PY on L1210 and Chinese hamster ovary cells were studied in relation to its intracellular localization and compared with the affinity of PY to bind to double-stranded DNA and RNA and its propensity to condense single-stranded DNA and RNA. Antitumor properties of PY were tested on L1210 leukemia and Sarcoma 180 ascites in mice. At a concentration of 1.7 to 3.3 microM, PY was localized almost exclusively in mitochondria of cultured cells, similar to another mitochondrial probe, rhodamine 123. At that concentration PY was not toxic but suppressed cell growth, arresting cells in G1. At a concentration of 6.7 to 33.0 microM, PY was also localized in nucleoli and uniformly in cytoplasm, bound to the RNase-sensitive material therein. At that high concentration PY induced cell arrest in G2 and S and was cytotoxic. The dye exhibited a propensity to bind and condense (precipitate) single-stranded nucleic acids, and condensation could be measured by the appearance of light-scattering products. Among a variety of natural and synthetic nucleic acids the most sensitive were the RNA polymer, polyriboadenylate, and the copolymer, polyriboadenylate and polyriboguanylate, which underwent condensation at a PY concentration of 6.6 to 10.0 microM. Natural and synthetic DNA polymers were resistant to condensation. The data suggest that the cytostatic (G2 and S arrest) and cytotoxic (inability to exclude trypan blue, loss of clonogenicity) effects of PY seen at 6.7 to 33.0 microM concentration may be a consequence of the dye binding to RNA. PY may intercalate to double-stranded RNA and/or cause the specific condensation of single-stranded RNA; the polyadenylated sections of mRNA appear to be the most sensitive cellular targets to undergo condensation. PY showed antitumor properties extending survival of L1210 leukemic mice by 50% and slowing growth of Sarcoma 180 ascites tumor. The possibility that certain antitumor drugs, generally believed to act via intercalation to DNA, may exert chemotherapeutic effects via their interactions with RNA is discussed.

Animals

[Possibility of spectrophotometric registration of conformation changes of polynucleotides in the system poly(A:U)-pyronine G].

The spectrophotometric study of poly(A:U) (90:10)-pyronine G (PG) complex formation has shown that the interaction of PG with polyriboadenylic acid pH = 6.3 is not changed despite the presence of 10% of pyrimidine bases in the single-stranded polynucleotide chain. Analysis of the dependence of complex formation versus acidity increase in the visible spectral range suggests that there are two forms of protonated double-stranded helix of poly(A:U) (90:10), as well as poly(A). PG dye does not interact with the more strongly protonated form by means of the aggregation. There is a strong resemblance between the interaction of PG with the less strongly protonated form and with the neutral single-stranded form of poly(A:U) (90:10). One can determine pH-transition ranges of the two poly(A:U) (90:10) protonated forms using the difference in their interaction with PG at low concentrations of polynucleotide.

Hydrogen-Ion Concentration

Photosensitizing effects of the tricyclic heteroaromatic cationic dyes pyronin Y and toluidine blue O (tolonium chloride).

Pyronin Y [3,6-bis(dimethylamino)xanthylium chloride; PY] and toluidine blue O [tolonium chloride; 3-amino-7-(dimethylamino)-2-methyl phenothiazin-5-ium chloride; TB] are cationic dyes commonly used in cytochemistry that have affinity to nucleic acids, predominantly to RNA. In live cells these dyes accumulate in mitochondria and sensitize the cells to light. The photosensitizing effects of PY and TB were compared with those of another mitochondrial cationic dye, rhodamine 123, and a noncationic dye, merocyanine 540, which binds to the cell membrane. Ninety % reduction of clonogenicity of human epidermoid carcinoma (A-253) cells pretreated with 3.3 microM PY, 0.67 microM TB, 13 microM rhodamine 123, or 18 microM merocyanine 540 was achieved by cell exposure to 0.7, 1.0, 1.2, or 1.5 J/cm2 doses of white light, respectively. The above concentrations of PY, TB, or merocyanine 540 represent the maximal ones at which the effect of each of these dyes alone, in the dark, in reducing cell clonogenicity was less than 12%. Exposure of A-253 cells to light at doses reducing clonogenicity by 50% caused a transient (24 h) arrest of the surviving cell population in the G1 phase of the cell cycle. In contrast to A-253 cells, Chinese hamster ovary cells were highly resistant to the photosensitizing effects of each of the four dyes. Also, the normal human lung fibroblasts (WI-38) were highly resistant to photosensitization by PY, whereas the simian virus 40-transformed WI-38 cells and another carcinoma line (OV-3) were sensitive. The data suggest that PY and TB, like other mitochondrial dyes, may have a selective antitumor photosensitizing activity.

Cell Cycle

[Spectrophotometric complexes of nucleic acids with pyronine G dyes as a test for radiation damage of DNA].

The spectrophotometric titration curves of the complexes of polynucleotide matrix with pyronine G are highly specific depending on the type and conformation of polyelectrolyte (native and denatured DNA, polyribonucleotides, tRNA). It is found that DNA from the liver of experimental animals of different age, permanent inhabitants of the Chernobyl zone, forms complexes with RNA. A method for testing the defects in a DNA secondary structure have been developed based on comparative analysis of the spectra for the complexes of different nucleic acids with dyes and relative curves of spectrophotometric titration.

Animals

Fixation of mammalian tissues in different fixatives and its influence on the staining with methyl green-pyronin.

This investigation is a study on the role of fixation of different mammalian tissues in different fixatives as well as in 10% neutral formalin containing different metal cations and its effect on the staining with the dye-mixture, methyl green-pyronin. The results indicate variation in the colour of the nuclei in tissues fixed in the different fixatives. The possible role of the fixatives on nuclear colouration has been discussed.

Animals