Effect of Acriflavine-Guanosine Compound (AG60) Treatment on the Gastrointestinal Endocrine Cells of Mouse Inoculated with Ehrlich Carcinoma Cells / 대한해부학회지

To study the tumor-suppression effect of a newly developed anti-tumor agent AG60 [ acriflavine (1) : guanosine (1) composition, Taerim Pharm. Co., Seoul, Korea], each Ehrlich carcinoma (107 cells)-inoculated mouse received the subcutaneous injection of 0.2 ml of saline, 5mg/kg of AG60, and 30 mg/kg of AG60, every other day for two weeks. Animals were sacrificed, and stomach, duodenum, appendix vermiformis and rectal tissues were resected and fixed in 10% neutral formalin. Tissue blocks were washed, dehydrated, embedded and cut in 6 microgram-thick sections. For immunocytochemistry, the streptavidine-biotin-peroxidse method was used with a InnoGenex (San Ramon, Calif., USA) staining kit. The tissues were incubated with rabbit antisera against somatostatin (Biogenesis, Poole, England, UK) diluted 1 : 300, secretin (Biogenesis, Poole, England, UK) diluted 1 : 2,400, neurotensin (Biogenesis, Poole, England, UK) diluted 1 : 2,600, or motilin (Biogenesis, Poole, England, UK) diluted 1 : 1,000 for 24 hour at 4dreeges C, followed by incubation in biotinylated antirabbit IgG and horseradish peroxidase-streptavidin conjugate for 1 hour at room temperature. The antigen-antibody reaction sites were visualized by incubating the sections with diaminobezidine tetrahydrochloride (DAB) for 5~15 minutes at room temperature. After mounting in canada balsam, they were examined in a Leica DM RB microscope. The number of the immunoreactive cells in the area of gastrointestinal mucosae (mean number of immunoreactive cells per 0.25mm2) were observed and calculated. The results are as follows : 1. In the fundic gland of normal mouse, somatostatin immunoreactive cells were detected (18.5+/-0.71), but neurotensin, secretin, or motilin immunoreactive cells were not found. In the duodenal mucosa of normal mouse, somatostatin immunoreactive cells were detected (7.0+/-0.10), but neurotensin, secretin or motilin immunoreactive cells were rarely found. 2. Immunoreactivity of somatostatin, secretin, neurotensin or motilin cells was not found in appendix vermiformis and rectum of normal mouse. 3. On immunocytochemical study, somatostatin immunoreactive cells in the fundic glands of normal, experimental control, AG60 (5mg/kg)-treated, AG60 (30 mg/kg)-treated and 5-fluorouracil (60 mg/kg)-treated groups were 18.5+/-0.71, 10.0+/-4.20, 11.5+/-0.71, 13.5+/-2.10, 11.5+/-2.71, respectively. 4. On immunocytochemical study, somatostatin immunoreactive cells in the duodenal mucosae of normal, experimental control, AG60 (5 mg/kg)-treated, AG60 (30 mg/kg)-treated and 5-fluorouracil (60 mg/kg)-treated groups were 7.0+/-2.10, 0.5+/-2.71, 3.0+/-1.41, 0.5+/-0.71, 2.50+/-0.71, respectively. 5. On immunocytochemical study, secretin immunoreactive cells in the duodenal mucosae of normal, experimental control, AG60 (5 mg/kg)-treated, AG60 (30 mg/kg)-treated and 5-fluorouracil (60 mg/kg)-treated groups were rarely found. 6. On immunocytochemical study, neurotensin and motilin immunoreactive cells in the duodenal mucosae of normal groups were detected, but immunoreactivies were not detected in experimental control, AG60 (5 mg/kg)-treated, AG60 (30 mg/kg)-treated or 5-fluorouracil (60 mg/kg)-treated groups.

Morphological Study on the Thymic Cortex of the Ehrlich Carcinoma-Inoculated Mice, Treated with 5-Fluorouracil and Acriflavine-Guanosine Complex (AG60) / 대한해부학회지

In cancer therapy, immunological disorder is one of most severe problem. Since thymic cortex is the home of T-cell proliferation and "education", thymic morphology following administration of certain drugs can be used as a parameter of immunological safety of the drug. In this study, morphology of thymic cortex, following administration of 5-fluorouracil or AG60, was studied. AG60 is a newly developed anti-cancer remedy, the compound of acriflavine and guanosine (1 : 1). ICR mice were subcutaneously inoculated with Ehrlich carcinoma cells (10(7) cells/mouse) in their inguinal areas. Each mouse in 5-fluorouracil group was injected subcutaneously with a single dose of 30 mg/kg of 5-fluorouracil every other day, and the mouse in AG60 group, with 30 mg/kg of AG60 (Taerim Pharm. Co., Seoul) every other day. The control mouse was injected with saline. The mice were sacrificed on the day after 7th injection. Tissues of thymic cortices were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde solution (0.1 M Millonig's phosphate buffer, pH 7.3), and refixed in 2% osmium tetroxide solution (0.1 M Millonig's phosphate buffer, pH 7.3). Tissue blocks were dehydrated, and were embedded in araldite mixture. For the overview-comparison, semithin sections stained with toluidine blue solution were photographed. And the typical portions were cut with ultratome, stained and observed with electron microscope. In light microscopy, thymic cortical morphology of AG60-injected mouse was similar with that of control mouse. But the cortical morphology of 5-fluorouracil-injected mouse was impressively different from those of the control or AG60 group mice. Thymocytes in the thymic cortex of 5-fluorouracil-injected mice were severely depleted. In electron microscopy, thymocytes in the thymic cortices of the control or AG60 group mice were crowded, and small groups of thymocytes were surrounded by the cytoplasmic processes of epithelial reticular cells. Mitotic figures were randomly seen. Thymocytes of 5-fluorouracil-injected mouse were naked out from the epithelial reticular cells, and were completely depleted out from the cortex composed mainly of enlarged epithelial reticular cells. Numerous microvilli were protruded from the naked thymocytes. The results were interpreted as that 5-fluorouracil induce leukopenia, and homing of lymphocytes to thymic cortex is severely depressed. 5-fluorouracil also disturb the normal protective and supportive function of epithelial reticular cells for thymocytes. Whereas the complex of acriflavine-guanosine compound (AG60) is immunologically safe, as seen in thymic cortical morphology.

Effects of BCG or AG60 on Gastric Parietal Cells of the Mouse Implanted with Ehrlich Carcinoma Cells / 대한해부학회지

This experiment was performed to evaluate the morphological responses of the gastric parietal cells of mouse inoculated with Ehrlich carcinoma cells, following administration of Bacillus Calmette -Guerin (BCG) or acriflavine -guanosine composition (AG60, Taerim Pharm. Co. Seoul, Korea). In the experimental groups, each mouse was inoculated with 1 X 10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day, 0.2 ml of saline, BCG (0.03 X 10(8) ~0.32 X 10(8) CFU) or AG60 (30 mg/kg) was injected subcutaneously to the animals every other day. Animals were sacrificed on the 14th day from the first injection. Pieces of the tissue were taken from the stomach, prefixed with 2.5% glutaraldehyde -1.5% paraformaldehyde, followed by post -fixation with 1% osmium tetroxide. The ultrathin sections stained with uranyl acetate and lead citrate were observed with a JEM 100CX -II electron microscope. In the experimental control, the BCG and the AG60 treated groups, most parietal cells showed reduced lumenal spaces of the intracellular canaliculi, since microvilli of intracellular canaliculi were very irregularly shaped and crowed with each other. And in the BCG and the AG60 treated mice, myelin figures, lysosomes and multivesicular bodies in the parietal cells were observed more frequently than in those of the experimental control ones. In the BCG treated rats, membranes of the tubulovesicles of the parietal cells were disintegrated, but the similar changes were not observed in the AG60 treated mice,. Above results suggest that the BCG treated animals inoculated with Ehrlich carcinoma cells might suffer from reduced acid secretion of the parietal cell, since the disintegration of the tubulovesicular membranes in the parietal cells are occurred following injections. Whereas AG60 dose not affect remakably defect on the parietal cells.

Effects of Adriamycin or AG60 to the DNA Synthesis of Duodenal Epithelium of Mice Implanted with Ehrlich Carcinoma Cells: An Autoradiographic Study / 체질인류학회지

This experiment was performed to evaluate the morphological responses of the intestinal gland of the mouse, duodenum inoculated with Ehrlich carcinoma cells, following administration of adriamycin or acriflavine -guanosine composition (AG60, Taerim Pharm. Co. Seoul, Korea). Healthy adult ICR mice weighing 25 g each were divided into normal and experimental groups (experimental control group, adriamycin treated group, and AG60 treated group). In the experimental groups, each mouse was inoculated with 1 x10 7 Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day, 0.2 ml of saline, adriamycin (2 mg/ kg), AG60 (5 mg/kg) or AG60 (30 mg/kg) were injected subcutaneously to the animals every other day, respectively. The day following the 7th injection of anticancer drugs, each mouse was injected with a single dose of 0.7 microCi/gm of methyl -3 H -thymidine (25 Ci/mmol, Amersham Lab., England) through tail vein. Seventy minutes after the thymidine injection, animals were sacrificed. The number of the labeled epithelial cells of the duodenal crypts (mean number of labeled epithelial cells per 3.5 mm length of mucosa) were observed and calculated. On histological study, in the duodenum of adriamycin treated groups, vesiculated epithelial cells of the intestinal villi, expanded lumen of the intestinal gland (G) and loosely arranged lamina propria were observed. But in the AG60 treated group, morphological changes of the duodenum were not observed. On autoradiographic study, number of the labeled cells of normal control, experimental control, adriamycin -treated, AG60 (5 mg/kg)-, and AG60 (30 mg/kg)-treated groups were 595.3 +/-48.96, 715.+/-89.11, 96.0 +/-15.62, 632.0 +/-83.16 and 370.3 +/-49.65, respectively. In the adriamycin and AG60 30mg/kg -treated group, poorly -labeled cells containing only a few silver grains of 3 H -thymidine were observed more frequently than in those of the normal control group. But in the experimental control group, number of the heavy labeled cells were observed more frequently than in those of the normal control group. From the above results, adriamycin and AG60 (30 mg/kg) may suppress the DNA synthesis of the cells of the duodenal crypts. But AG60 does not result any histological defect on the duodenal mucosa. These results suggest that AG60 is expected as one of most effective anticancer drugs.

Anticancer Effect of AG60 (Acriflavine-Guanosine Compound) on the Ehrlich Cancer Cells Light Microscopic, Autoradiographic and Electron Microscopic Study / 대한해부학회지

To evaluate the effect and working mechanism of a newly developed anti-cancer drug, AG60 (acriflavine-guanosine compound, Taerim Pharm. Co. Seoul, Korea), histotologic, autoradiographic and electron microscopic studies were carried out. For the histologic study, each Ehrlich carcinoma cells (10(7) cells)-inoculated mouse was subcutaneously injected with saline (0.2 ml), 10 mg/kg of AG60, or 30 mg/kg of AG60, every other day, respectively. Animals were sacrified on the 14th day from the first injection, and tumor masses were fixed in 10% formalin solution. Tissue sections of the tumor were stained with hematoxylin and eosin. For the electron microscopic study, Ehrlich carcinoma (10(7) cells)-inoculated mice were subcutaneously injected every other day with saline (0.2 ml) or 30 mg/kg of AG60, respectively. The day after 7th injection (14th day), animals were sacrified, small piece of tumor masses were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde solution followed by fixation in 2% osmium tetroxide solution. Ultrathin sections were counter stained with uranyl acetate-lead citrate solutions, and observed with JEM 100CX electron microscope. For the autoradiographic study, each Ehrlich carcinoma (10(7) cells)-inoculated mouse was injected every day with 0.2 ml of saline, 5 mg/kg of AG60, or 30 mg/kg of AG60, respectively. The day following the last injection, each animal was given a single dose of 0.7 micricurie/g of methyl-3H-thymidine (Amersham Lab., England) through the tail vein. Seventy minutes after the thymidine injection, animals were sacrified, tumor masses were collected and fixed in 10% neutral formalin. Tissue blocks were washed, dehydrated, embedded and cut in 6 micrometer-thick sections. Deparaffinzied sections were dipped in the autradiographic emulsion E1 (Amersham Lab., England) and dried and stocked in the dark room. Filmed sections were exposured five weeks in the dark room, and were developed in the developer. Labeled indices (mean number of labeled cells per 100 cancer cells) and labeled grain indices (mean number of labeled silver grains per one cancer cell, and total granule numbers per every 100 cancer cell) were observed and calculated. The results were as follows : 1. On histological study, massive apoptosis were occured following the injection of AG60. Only small number of live cancer cells were observed. 2. On electron microscopic study, massive apoptotic figures including fragmentation of nuclei and cytoplasms, multiple nucleoli, condensation of nucleus and cytoplasm, deep invaginations and microcleft formations of nuclei, margination of heterochromatin along the inner nuclear membrane and microcleft , etc. were noticed. Giant cells represent the "tumor cell-tumor cell emperipolesis", and many of them seem to be in process of "cytolytic emperipolesis". 3. On autoradiographic study, labeled grains of 3H-thymidine were suppressed to only 11%~5% of control cancer cells following AG60 administrations. Discussed on the above experiments, it is suggested that severe suppression of DNA, RNA and protein syntheses by AG60 induce massive apoptosis of cancer cells. AG60 is expected as one of most effective anticancer drugs for the cytostatic therapy, the disease stabilization, the improved quality of life, the prolongation of life, and possibly the chemoprevention.

Fluorescent Study on the Tissue-Distribution of Acriflavine-Guanosine Compound (AG60) / 대한해부학회지

The study was carried out to evaluate the tissue-distribution of acriflavine or AG60 (acriflavine-guanosine compound, 1 : 1), the newly developed anticancer remedy. Successful access or distribution of a drug to specific tissue is important to attack the cancer cells in the same area. But it also means that the drug may disturb the activities of labelled tissues or cells. On the other hand, unlabelled elements may survive from massive treatment with the drug. In this study, distribution of acriflavine or AG60 in Yac-1 leukemic cells (0.25~25 microgram/ml) and in the tissues of rats or mice (5~50 mg/kg) were evaluated. Yac-1 cells showed prominent fluorescence on the heterochromatin and more or less prominent fluorescence on the nucleoplasm, cytoplasm and plasma membrane. Cytotoxicity of AG60 led to morphologic changes such as bleb- or baloon-formation on the surface, general swelling of the cell, and lysis of the cell. Following the subcutaneous administration of acriflavine or AG60 (5~50 mg/kg) to the Ehrlich carcinoma-inoculat-ed rats or mice, most tissues including cancer cells showed acriflavine-fluorescence with some exception. The nuclei of cells of tissues were labelled more strongly than those of cytoplasm. Fluorescence was especially strong over biliary tree, renal corpuscle, gastrointestinal mucous coat, and bronchial mucous coat. But parenchymal components of central nervous system did not show any fluorescence. As shown in Yac-1 cells treated with AG60, the drug strongly attached to nucleic acids, and it induced swelling and disintegration of cancer cells. Fast turn-over of AG60 was seen in the secretory passages of bile juice, urine, gastrointestinal mucin, and bronchial mucin. The results show that AG60 could reach most tissues except parenchymes of central nervous system.

Anti-Tumor Effect of AG60 against Ehrlich Tumor

BACKGROUND: AG60 is a complex of acriflavine and guanosine. Our previous study revealed that AG60 had not only in vitro antitumor activities in several human cancer cell lines, but also strong antitumor effects in animal experiments using p388 or S180 cells-implanted mice. METHODS: Antitumor effects of AG60 were compared with those of Adriamycin, acriflavine, guanosine or control group. Body weight, tumor weight change, and survival time were measured in Ehrlich carcinoma cells implanted ICR mice. RESULTS: Body weights in AG60, acriflavine, or Adriamycin treated groups were significantly lower than those in control group during 30 day observation period(p<0.05). The percent tumor growth inhibition of AG60, Adriamycin, acriflavine, or guanosine two weeks after last treatment was respectively 86% (T/C%=14), 83% (T/C%=17), 68%(T/C%=32), 41% (T/C%=59). According to above data, tumor growth inhibition in AG60 treated group was significantly stronger than that in control, acriflavine or guanosine treated group(p<0.01), but there was no significant difference between AG60 and Adriamycin treated group. Mean survival time in control, AG60, Adriamycin, acriflavine, or guanosine treated group was respectively 33+/-3.9 days, 68+/-4.2 days, 54+/-5.8 days, 36+/-3.8 days, 50+/-8.1 days. CONCLUSIONS: The anti-tumor effect of AG60 against Ehrlich tumor was significantly stronger than that of control, acriflavine or guanosine, and comparable with Adriamycin. Mean survival time in AG60 treated group was significantly longer than that in control, acrifavine, guanosine or Adriamysin treated group.

Influencia de diferentes derivados lácteos sobre el crecimiento de Listeria monocytogenes en medios selectivos / Influence of different dairy products on the growth kinetic of Listeria monocytogenes

Se estudió la influencia del añadido de crema de leche y leche parcialmente descremada sobre la cinética de crecimiento de Listeria monocytogenes en caldos de enriquecimiento para listerias, conteniendo diferentes concentraciones de acriflavina (15 y 7,5 mg/l). El crecimiento de Listeria monocytogenes en los caldos de enriquecimiento sufrió un retardo atribuible, al menos parcialmente, a la presencia de acriflavina. El añadido de crema de leche o leche parcialmente descremada al caldo de enriquecimiento que contiene 7,5 mg/l de acriflavina produjo un alargamiento de la fase de adaptación, pero las cosechas máximas alcanzadas a las 48 h no mostraron diferencias significativas. En presencia de 15 mg/l de acriflavina, se observó una pérdida inicial de la viabilidad de los cultivos, que fue potenciada por el agregado de crema de leche o leche parcialmente descremada al caldo de enriquecimiento. Además, la leche descremada produjo una disminución de la velocidad máxima de crecimiento que impidió alcanzar la cosecha máxima dentro de las 48 h. Los resultados obtenidos sugieren la necesidad de validar la metodología de recuperación de L. monocytogenes para cada producto, pues la eficiencia de recuperación podría ser afectada por la composición del mismo, sobre todo cuando la carga microbiana es baja

Anti-tumor Effect of the Complex of Acriflavine and Guanosine (AG60) / 대한암학회지

PURPOSE: The anti-tumor effect of the complex of acriflavine and guanosine (AG60) was investigated. MATERIALS AND METHODS: In vitro cytotoxicity of AG60 was measured using SRB assay, and in vivo antitumor activity of AG60 was examined in CDF1 mice intraperitoneally inoculated with the P388 leukemic cells and in ICR mice inguinally implanted with S-180 cells. Tumor size and mean survival time were determined. RESULTS: AG60 and acriflavine showed strong anti-tumor effect in vitro on lung cancer (A549), renal cancer (UO-31) and colon cancer (COLO205) cells. However, AG60 did not show the cytotoxicity against normal cell line, 3T3. The range of the IC50 of AG60 to the various tumor cell lines was 0.09 microgram/ml through 1.94 microgram/ml. The treatment of ascitic tumor bearing CDF1 mice with AG60 resulted in over 160% increases in the mean survival time. The most effective dose of AG60 was 30 mg/kg body weight in tumor implanted mice. In solid tumor bearing ICR mice tumor growth and progression were suppressed in response to the different doses at 30 days; 69.8% suppression of tumor size in response to acriflavine, 16.0% to guanosine, 87.7% to AG60 and 78.5% to doxorubicin. In addition, 35% increases were observed in the means survival time of AG60 treated group compared with control group. CONCLUSION: The prominant anti-tumor effects of AG60 shown in this report would represent the possibility of the clinical trials.

Método de rivanol modificado con acriflavina más oxitocina experiencias de 10 años / Methods of rivanol modified with acriflavina with `oxytocin` experiences of 10 years you

Se revisan las 377 historias clínicas de mujeres a las que se le realizó la interrupción de la gestación en el segundo trimestre por el método de Rivanol, modificado con acriflavina más oxitocina, en el decenio 1980-1989, donde predominaron las mujeres con 20 años o menos que eran primigestas y nulíparas. En este grupo de edades predominaron las causas sociales, mientras que hubo un alza de frecuencia por motivo de la vacuna anti-rubeólica. En los últimos años aumento el diagnóstico de las malformaciones fetales, las demás causas se mantuvieron estables en su frecuencia. El 92,4 por ciento de las pacientes expulsaron antes de las 48 horas y la mitad lo hizo antes de las 24 horas. No hubo diferencias significativa en el tiempo de expulsión, entre las que tenian menos de 18 semanas. El uso de la oxitocina constribuyó a disminuir el tiempo de expulsión. Hubo un 18,9 por ciento de complicaciones, donde predomiron la fiebre y los sangramientos; presentaron un 1,32 por ciento de pacientes complicados de gravedad extrema. La utilización de este método es una alternativa al Rivanol

Mutagenesis of candida albicans by N-Methyl N-Nitro-N-Nitroso guanidine, ultraviolet and acriflavine

N-methyl-N-nitro N-nitroso guanidine [MNNG], Acriflavine [A C] and Ultraviolet [UV] have been used to induce mutation in 4 clinical isolates of C. albicans. Sixteen stable mutants were isolated with MNNG, 11 with AC and 4 mutants with UV. The most lethal effect was recorded, with MNNG [99.48%] and the least lethal action was with UV [77.70%]. UV gives the highest percentage of revertant [73.3%]. The results showed marked differences between the 4 strains to the mutagenic effects of MNNG, AC and UV. These differences could be attributed to differences in the genetic background of the four strains

A theoretical study of acriflavin-DNA binding.

A theoretical study of binding behaviour of acriflavin, a well-known mutagen, with DNA base pairs such as AT, GC, TA and CG has been performed using CNDO/2 method to compute net atomic charges and dipoles located at various centres in acriflavine as well as base pairs. Acriflavine-DNA base pair interactions have been evaluated using second order perturbation method with multicentered multipole approximation. Only minimum energy configurations have been reported. Results have been discussed with a view to obtain a comparative behaviour of other similar dyes like proflavine and acridine orange.

Efeitos da acriflavina em diferentes espécies de streptomyces produtores de actinomicina / Effects of acriflavine on three actionomycin producer species of Streptomyces

O tratamento de três diferentes espécies de Streptomyces com acriflavina, resultou na perda da produçäo de actinomicina em 5-12% das colônias isoladas nestas linhagens. S. felleus e S. regensis mostraram mais instabilidade do que S. parvulus. A produçäo de pigmento amarelo característica destas linhagens produtoras de actinomicina foi totalmente eliminada. A instabilidade genética destas linhagens pode ser atribuída a elementos extracromossômicos

Acao de alguns agentes curagenicos na perda da excrecao de protase em Proteus mirabilis. / Effects of some cu excretion of Proteus mirabilis

Drogas curagenicas, como brometo de etidio acriflavina e mitomicina C, aumentam grandemente, a conversao de celulas excretoras de protease instaveis de Proteus mirabilis em celulas nao excretoras. Esse efeito nao ocorre sobre celulas excretoras estaveis de protease. A rifampicina apenas seleciona celulas protease-negativas, por eliminacao preferencial de celulas excretoras.Temperaturas superiores a fisiologica nao sao efetivas na perda de excrecao de protease em linhagens de P. mirabilis que excretam protease de maneira instavel