Antioxidant, Metabolic And Antitumor Activity Of Triterpenoids Combination With Cytostatics

Objective: To study the effect of betulin derivatives combination with 5-fluorouracil or hydrazine sulfate on the ROS generation, the SOD and LDH activity using rat blood, as well as the effect of combination drugs on Ehrlich carcinoma in experiments on mice. Methods: We used a chemiluminescence technique to study the ROS generation, and spectrophotometry to determine the MDA level and the SOD and LDH activity. The model of transplanted Ehrlich ascites carcinoma was investigated on mice using a cytological analysis of ascitic fluid cells according to Pappenheim`s method. Results: In vitro experiments on rat blood at the doses of 2, 5 and 10 μg per ml revealed the dose-dependent effect of combination drugs on the antioxidant properties. In plasma, the ROS generation and the MDA level increased by 10-300% in comparison with control at the doses of 5 and 10 μg per ml only. Still, the SOD and LDH activity in general increased by 10-130% in comparison with control under the action of the studied combination drugs. The study on mice showed the effectiveness of a combination of triterpenoids and cytostatics in Ehrlich ascites carcinoma therapy. The state and behavior of the animals improved, the volume of ascites fluid decreased by 40-50% after treatment for 10 d. Conclusion: The combination of betulin derivatives with cytostatics can be used as antitumor drugs in Ehrlich ascites carcinoma therapy that is due to metabolic plasticity, increased ROS generation in enhanced antioxidant enzyme protection.

Effect of ketoprofen and indomethacin on methotrexate pharmacokinetics in mice plasmaand tumor tissues.

Methotrexate (MTX) has been used in combination with nonsteroidal antiinflammatory drugs (NSAIDs) in the treatment of inflammatory diseases and malignancies. Severe adverse effects with this combination may occur, usually resulting from inhibition of renal transporters. Solid Ehrlich Carcinoma was induced by implantation of Ehrlich Ascites Carcinoma (EAC) cells subcutaneously into the thigh of mice and after 30 days, mice were divided into 3 groups , Group I served as control group received MTX (50 mg/kg, i.p.), Group II received Ketoprofen (100 mg/kg, i.p.) then after half an hour received MTX (50 mg/kg, i.p.), Group III received Indomethacin (10 mg/kg, i.p.) then after half an hour received MTX (50 mg/kg, i.p.). Plasma and tissue samples were collected at different times then MTX concentrations were determined by HPLC. The injection of Ketoprofen or Indomethacin before MTX injection caused significant increase in the AUC and CPmax of MTX (p < 0.05) and significant decrease in CL/F and Vd/F of MTX (p < 0.05) in mice plasma. The study showed that administration of ketoprofen or indomethacin prior to MTX caused significant decrease in MTX elimination and significant increase in MTX extent of absorption which may lead to severe adverse effects if coadministered in human.

Effects of AG60 on Gastric Chief Cells of the Mouse Implanted with Ehrlich Carcinoma Cells / 대한해부학회지

This experiment was performed to evaluate the morphological responses of the gastric epithelial cells and the gastric chief cells of the mouse inoculated with Ehrlich carcinoma cells in the inguinal area following administration of acriflavine-guanosine composition (AG60). Healthy adult ICR mice were divided into normal and experimental groups. In the experimental groups, each mouse was inoculated with 1x10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. The day following the 7th injection of saline or AG60, each mouse was injected with methyl-3H-thymidine through tail vein. Seventy minutes after the thymidine injection, gastric tissues were taken and fixed in 10% buffered neutral formalin. Deparaffinized sections were coated with autoradiographic emulsion EM-1 and dried, and then placed in a light-tight box. The number of labeled epithelial cells in the gastric mucosae were observed and calculated. And for electron microscopic observation, gastric tissues were prefixed with 2.5% glutaraldehyde-1.5% paraformaldehyde solution, followed by post-fixation with 1% osmium tetroxide solution. The ultrathin sections were stained with uranyl acetate and lead citrate. The size of zymogen granules and mitochondria in the gastric chief cells were observed and calculated. On the autoradiographic study, number of labeled cells in the area of 3.5 mm width (6 micrometer thickness) of mouse gastric mucosae of normal control, tumor control and AG60-treated groups were 319.7+/-66.46, 343.7+/-47.72 and 102.3+/-54.99 respectively. On the electron microscopic study, the size of zymogen granule in the gastric chief cells of normal control, tumor control and AG60-treated groups were 0.74+/-0.208 micrometer, 1.18+/-0.291 micrometer and 0.97+/-0.259 micrometer, respectively. And the mitochondrial size of the gastric chief cells of normal control, tumor control and AG60-treated groups were 0.86+/-0.364 micrometer, 1.02+/-0.466 micrometer and 0.92+/-0.390 micrometer, respectively. And in the AG60 treated group, most chief cells did not show any difference in ultrastructure, except that myelin figures were more frequently observed, in comparison with that of nornmal control group. From the above results, AG60 may suppress the DNA synthesis of the gastric epithelial cells, but does not results severe fine structural defect on the gastric chief cells. These results suggest that AG60 is expected as one of the most effective anticancer drugs.

The Influence of Alpha-fetoprotein on Natural Suppressor Cell Activity and Ehrlich Carcinoma Growth

The influence of alpha-fetoprotein (AFP) on the bone marrow (BM) natural suppressor (NS) cells of intact Ehrlich carcinoma -bearing CBA mice was studied. Bone marrow NS cells were fractionated into three fractions by isopycnic centrifugation on percoll gradients: NS1 (rho=1.080 g/ml), NS2 (rho=1.090 g/ml) and NS3 (1.100>rho>1.090 g/ml). These fractions were highly different in their sensitivity to known NS cell inductors (interleukin (IL)-2, IL-3 or histamine). None of the NS fractions isolated from the intact mice spontaneously produced antiproliferative activity, however, they showed a high level of NS (antiproliferative and natural killer cell inhibitory) activity under the influence of AFP. A single injection of AFP to intact mice led to an increase of spontaneous NS activity and the inhibition of natural killer cell activity. NS activity, especially NS2, was increased in when tumor cells were subcutaneously inoculated three days after AFP injection. In the AFP-treated mice, the tumor mass at 14 days was 60% larger than that in the untreated mice. Our data confirmed that AFP is a tumor marker that can inhibit cancer immunity and plays a role in cancer pathogenesis.

Effects of BCG or CP-2 on the DNA Synthesis in the Epithelial Cells of the Mouse Appendix / 대한해부학회지

This experiment was performed to evaluate the morphological responses of the appendicular mucosa of the mouse, inoculated with Ehrlich carcinoma cells in the inguinal area, following administration of BCG or CP-2 (Coptis chinensis-Croton tiglium extracts). Healthy adult ICR mice weighing 25 gm each were divided into normal and experimental groups (experimental control, BCG or CP-2 treated group). Each experimental group mouse was inoculated with 1 x 10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. From the next day after inoculations, 0.2 mL of saline, BCG (0.5 mL/25 g B.W.: 0.03 x 10(8) ~ 0.32 x 10(8) CFU) or CP-2 (30 mg/kg) were injected subcutaneously to the animals every other day, respectively. The day following the 7 th injection of BCG or CP-2, each mouse was injected with a single dose of 0.7 microCi/gm of methyl-3H-thymidine (25 Ci/mmol, Amersham Lab., England) through tail vein. Seventy minutes after the tritiated thymidine injection, animals were sacrificed. The number of the labeled epithelial cells of the appendicular mucosae were observed and evaluated. On histological studies of the experimental control, BCG or CP-2 treated mice, general morphologies of the appendicular mucosae were similar. On autoradiographic study, number of the labeled cells of normal control, experimental control, BCG treated or CP-2 treated groups were 362.2+/-56.12, 350.7+/-42.65, 265.8+/-27.08 and 241.3+/-53.29, respectively. Above results show that BCG and CP-2 suppress the DNA synthetic activity of the epithelial cells of the appendix, but did not show any remarkable morphological alterations on the mucosae. These results suggest that BCG and CP-2 are ones of effective anticancer drugs for the cytostatic therapy.

Effects of Acriflavine-Guanosine Composition (AG60) on the DNA Synthesis and Ultrastructure of Epithelial Cells of the Appendix of Mice Inoculated with Ehrlich Carcinoma Cells / 대한해부학회지

This experiment was performed to evaluate the morphological responses of the mucosa of the mouse appendix, inoculated with Ehrlich carcinoma cells in the inguinal area, following administration of Acriflavine-Guanosine Composition (AG60). Healthy adult ICR mice weighing 25 gm each were divided into normal, experimental control and AG60 treated group. Experimental control and AG60 treated groups, mice were subcutaneously inoculated with 1 x 10(7) Ehrlich carcinoma cells in the inguinal area. From next day after the carcinoma cell inoculations, 0.2 mL of saline or AG60 (5 mg/kg/0.2 mL) were injected subcutaneously to the animals every other day, respectively. The day following the 7 th injection of saline or AG60, each mouse was injected with a single dose of 0.7 microCi/gm of methyl-3H-thymidine (25 Ci/mmol, Amersham Lab., England) through tail vein. Seventy minutes after the 3H-thymidine injection, animals were sacrificed, and appendix tissues were fixed in 10% formalin solution for light microscopy. The number of the labeled mucosal epithelial cells of the appendix were observed and evaluated. For the electron microscopic study, the tissues were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde solution, followed by post-fixation with 1% osmium tetroxide solution. Ultrathin sections were counter stained with uranyl acetate-lead citrate solutions, and observed. On light microscopic observation of experimental control and AG60 treated mice, did not show any remarkable morphological alterations on the mucosae. On autoradiographic study, number of the labeled cells within 3.5 mm width mucosae of normal control, experimental control, AG60 treated mice were 362.2+/-56.12, 350.7+/-42.65 and 90.7+/-33.48, respectively. On ultrastructural observation of the experimental control and AG60 treated mice, general morphologies of the epithelial cells of appendix were similar. But intranuclear filamentous structures, intramitochondrial dense granules, and myelin figures were occasionally observed in the absorptive cells of AG60 treated mice than control ones. Above results show that AG60 suppress the DNA synthetic activity of the mucosal epithelial cells of mouse appendix, but did show slight ultrastructural alterations in the absortive cells. These results suggest that AG60 is one of effective anticancer drug for the cytostatic therapy.

Effects of Antitumor Agents to the DNA Synthesis of Cecal Mucosa of Mouse / 체질인류학회지

This experiment was performed to evaluate the morphological responses of the cecal mucosa of the mouse, inoculated with Ehrlich carcinoma cells in the inguinal area, following administration of 5-fluorouracil, mitomycin C or adriamycin. Healthy adult ICR mice weighing 25 gm each were divided into normal and experimental groups. In the experimental groups, each mouse was inoculated with 1 x 10(7) Ehrlich carcinoma cells subcutaneous in the inguinal area. From next day, 0.2 mL of saline, 5-fluorouracil (30 mg/kg), mitomycin C (400 microgram/kg) or adriamycin (2 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 micro Ci/gm of methyl-3H-thymidine (25Ci/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 cecal crypts (mean number of labeled epithelial cells per 3.5 mm length of mucosa) were observed and evaluated. On histological study, in the experimental control and mitomycin C-treated mice, general morphology of the cecal mucosae was similar. And in the 5-fluorouracil-treated mice, slightly swelled epithelial cells and expanded lumen of the intestinal crypts were observed. But in the adriamycin-treated groups, slightly disrupted intestinal crypts, a large number of basophilic epithelial cells and the expanded lumen of the intestinal crypts were observed. On autoradiographic study, number of the labeled cells of normal control, experimental control, 5-fluorouracil treated, mitomycin C-treated, or adriamycin-treated groups were 362.2+/-56.12, 350.7+/-71.13, 215.7+/-80.55, 144.2+/-34.60 and 125.0+/-37.45, respectively. In the adriamycin and mitomycin C-treated groups, poorly-labeled cells containing only a few silver grains were observed more frequently than in those of the normal and experimental control groups. From the above results, adriamycin and mitomycin C suppressed the DNA synthesis of the epithelial cells of the cecal mucosa more severely as compared with 5-fluorouracil did. Especially, adriamycin was more harmful than mitomycin C and 5-fluorouracil on the cecal mucosae.

Effects of Antitumor Agents to DNA Synthesis in the Gastric Epithelia of Mice Implanted with Ehrlich Carcinoma Cells: An Autoradiographic Study / 대한해부학회지

This experiment was performed to evaluate the morphological responses of the gastric epithelium of the mouse, inoculated with Ehrlich carcinoma cells, following administration of 5-fluorouracil, adriamycin or mitomycin C. Healthy adult ICR mice weighing 25 gm each were divided into normal control and experimental groups (tumor control group, 5-fluorouracil treated group, adriamycin treated group, and mitomycin C treated group). In the experimental groups, each mouse was inoculated with 1 x 10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day after inoculations, 0.2 mL of saline, 5-fluorouracil (30 mg/kg), adriamycin (2 mg/kg) or mitomycin C (400 microgram/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-3H-thymidine (25Ci/mmol, Amersham Lab., England) through tail vein. Seventy minutes after the thymidine injection, animals were sacrificed. The number of labeled epithelial cells in the gastric mucosae (mean number of labeled epithelial cells per 3.5 mm length of mucosa) were observed and calculated. On histological study, in the gastric mucosae of adriamycin-treated groups, denatured surface epithelial cells, expanded lumen of the gastric gland, and congested lamina propria were observed. But in the 5-fluorouracil or mitomycin treated groups, severe morphological changes of the gastric mucosae were not observed. On autoradiographic study, numbers of the labeled cells in the gastric mucosae per 3.5 mm length of normal control, tumor control, 5-fluorouracil-treated, adriamycin-treated and mitomycin C treated groups were 267.3 (+/-48.86), 273.6 (+/-59.41), 375.3 (+/-83.36), 15.3 (+/-9.66) and 124.0 (+/-32.66), respectively. In the adriamycin and mitomycin C-treated group, poorly-labeled cells containing only a few silver grains of 3H-thymidine were observed more frequently as compared in those of the normal control group. But in the 5-fluorouracil-treated 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 mitomycin C may severely suppress the DNA synthesis of the epithelial cells of the gastric mucosae. But some amount of the 5-fluorouracil (30 mg/kg) may not suppress the DNA synthesis of gastric epithelial cells.

Effects of Adriamycin or CP-2 to the Rectal Mucosae of Mouse Implanted with Ehrlich Carcinoma Cells: An Autoradiographic Study / 대한해부학회지

This experiment was performed to evaluate the morphological responses of the rectal intestinal glands of the mouse, inoculated with Ehrlich carcinoma cells, following administration of adriamycin or composition of the extracts of the Croton tiglium and Coptis chinensis rhizome (CP-2, Institute of Experimental Tumor Research, Seoul, Korea). Healthy adult ICR mice weighing 25 gm each were divided into normal and experimental groups (tumor control group, adriamycin treated group, and CP-2 treated group). 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, adriamycin (2 mg/kg) or CP-2 (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- 3H-thymidine through tail vein. Seventy minutes after the thymidine injection, animals were sacrificed. and rectal tissues were collected and fixed in 10% neutral formalin. Deparaffinized sections were coated with autoradiographic emulsion EM-1 (Amersham Lab., England) in the dark room and dried. The number of the labeled epithelial cells of the rectal crypts (mean number of labeled epithelial cells per 3.5 mm length of mucosa) were observed and calculated. On histological study, in the rectum of adriamycin treated groups, length of the intestinal crypts is shorter than those of the normal control ones. Disrupted intestinal crypts and epithelial cells were observed. But in the CP-2 treated group, morphological changes of the rectum were not observed. On autoradiographic study, number of the labeled cells of normal control, rumor control, adriamycin-treated, CP-2-treated groups were 263.1 (+/-38.65), 395.7 (+/-52.52), 73.3 (+/-22.54), 96.3 (+/-28.36), respectively. In the adriamycin and CP-2 treated groups., poorly-labeled cells containing only a few silver grains of 3H-thymidine were observed more frequently than in those of the normal and tumor control groups. But in the tumor 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 CP-2 may suppress the DNA synthesis of the cells of the rectal crypts. But CP-2 does not result any histological defect on the rectal mucosa. These results suggest that CP-2 is expected as one of most effective anticancer drugs.

Ultrastructural Alterations in the Gastric Chief Cells of Mouse, induced by 5-Fluorouracil or Mitomycin C / 대한해부학회지

This experiment was performed to evaluate the morphological responses of the gastric chief cells of the mouse, inoculated with Ehrlich carcinoma cells in the inguinal area, following administration of 5-fluorouracil or mitomycin C. Healthy adult ICR mice weighing 25 gm each were divided into normal and experimental groups (experimental control group, 5-fluorouracil-treated group and mitomycin C-treated group). In the experimental group, 1x107 Ehrlich carcinoma cells were inoculated subcutaneously in the inguinal area. From next day after inoculations, 0.2mL of saline (experimental control group), 5-fluorouracil (30 mg/kg, 5-fluorouracil-treated group), or mitomycin C (400 microg/kg, mitomycin C-treated group) were injected subcutaneously to the animals every other day, respectively. The day following the 7th injection, animals were sacrificed. Pieces of the tissue were taken from the gastric mucosa, prefixed with 2.5% glutaraldehyde-1.5% paraformaldehyde solution, followed by post-fixation with 1% osmium tetroxide solution. The ultrathin sections were stained with uranyl acetate and lead citrate. The size of zymogen granule and the size of the mitochondrion in the gastric chief cells were observed and compared. In the 5-fluorouracil treated group, most chief cells did not show any difference in ultrastructure, except myelin figures were more frequently observed, in comparison with those of normal control group. But in the mitomycin Ctreated group, necrotic cells were more frequently observed than in normal control and 5-fluorouracil-treated group. The size of zymogen granule in the gastric chief cells of normal control, experimental control, 5-fluorouracil-treated and mitomycin C-treated groups were 0.98 (+/-0.108)microm, 1.05 (+/-0.092)microm, 0.94 (/-0.123)microm and 0.93 (+/-0.156)microm, respectively. And the size of mitochondrion in the gastric chief cells of normal control, experimental control, 5-fluorouracil-treated and mitomycin C-treated groups were 0.80 (+/-0.130)microm, 0.83 (+/-0.143)microm, 0.87 (+/-0.165)microm and 0.81 (+/-0.083)microm, respectively. From the above results, in the treatment of low therapeutic doses of anticancer drugs into the animals inoculated with Ehrlich carcinoma cells, 5-fluorouracil may not suppress function of the gastric chief cells, but mitomycin C may exert a vicious influence on the function of the gastric chief cells.

Effects of 5-Fluorouracil, Mitomycin C or AG60 to the DNA Synthesis of Rectal Epithelium of Mice Implanted with Ehrlich Carcinoma Cells / 체질인류학회지

This experiment was performed to evaluate the morphological responses of the intestinal gland of the mouse, rectum inoculated with Ehrlich carcinoma cells, following administration of 5- fluorouracil, mitomycin C or AG60. Healthy adult ICR mice weighing 25 gm each were divided into normal and experimental groups (tumor control group, 5-fluorouracil, mitomycin C treated group, and AG60 treated group). In the experimental groups, each mouse was inoculated with 1*10 (7) Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day, 0.2 mL of saline, 5-fluorouracil (30 mg/kg), mitomycin C (400 microgram/kg) or AG60 (5 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-3H-thymidine through tail vein. Seventy minutes after the thymidine injection, animals were sacrificed. The number of the labeled epithelial cells of the rectal crypts (mean number of labeled epithelial cells per 3.5 mm length of mucosa) were observed and calculated. On histological study, in the rectum of mitomycin C treated groups, narrowed intestinal gland, a number of the nectotic changed epithelial nuclei and loosely arranged lamina propria were observed. But in the AG60 treated group, morphological changes of the rectum were not observed. On autoradiographic study, number of the labeled cells of normal control, tumor control, 5-fluorouracil (30 mg/kg) treated, mitomycin C (400 microgram/kg) treated and AG60 (5 mg/kg) treated groups were 246.3+/-42.30, 253.8+/-20.54, 172.7+/-19.02, 108.7+/-17.67 and 53.8+/-11.70, respectively. In the AG60 and mitomycin C treated group, poorly-labeled cells containing only a few silver grains of 3H-thymidine were observed more frequently than in those of the normal control group. From the above results, AG60 (5 mg/kg) and mitomycin C (400 microgram/kg) are more suppressed the DNA synthesis of the cells of the rectal crypts as compare with 5- fluorouracil (30 mg/kg). And AG60 does not result any histological defect on the rectal mucosa. These results suggest that AG60 is expected as one of most effective anticancer drugs.

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 Bacillus Calmette-Guerin (BCG) on the Spleen of Mouse Inoculated with Ehrlich Carcinoma Cells: A Morphological Study / 대한해부학회지

This experiment was performed to study the morphological responses of the spleen of mouse inoculated with Ehrlich carcinoma cells, following administration of Bacillus Calmette-Guerin (BCG). Healthy adult ICR mice weighing 25 g each were divided into normal and experimental groups (experimental control group and BCG treated group). In the experimental groups, each mouse was inoculated with 1X10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day, 0.2 ml of saline or BCG (0.03X10(8)-0.32X10(8) CFU) were injected subcutaneously to the animals every other day. The day following the 7th injection, each mouse was injected with a single dose of 0.7 microCi/gm of methyl-3H-thymidine (25 Ci/mmol, Amersham Lab., England) through tail vein. Seventy minutes after the 3H-thymidine injection, animals were sacrificed. Pieces of the splenic tissue, fixed in 10% neutral formalin for light microscopy. The sections were coated with autoradiographic emulsion EM-1 (Amersham Lab., England) and the coated sections were exposured for 5 weeks in the dark room. For electron microscopy, tissues were prefixed with phosphate buffered 2,5% glutaraldehyde-1.5% paraformaldehyde (pH 7.3), and post-fixed with phosphate buffered 1% osmium tetroxide solution (pH 7.3). Ultrathin sections of the white pulp area stained with uranyl acetate and lead citrate were observed with a JEM 100CX-II electron microscope. On histological study in the splenic white pulp, BCG treated mice showed more macrophages containing pyknotic nuclei than normal or experimental control mice showed. On autoradiographic study, a large number of the 3Hthymidine labeled cells were seen near the marginal zone, whereas only a small number of labeled cells were seen in the red pulp or the white pulp of the spleen. The number of the labeled cells in experimental control group was similar to that in the normal control mice, whereas that in BCG-treated mice was significantly increased as compared with that of normal control one. On electron microscopic study, in the white pulp of BCG treated mouse, mitotic cells were observed more frequently than in those of the normal or experimental control mice. In the BCG treated mice, macrophages and plasma cells in the white pulp were observed more frequently than in those of the normal or experimental control mice, whereas a few eosinopile leucocytes were observed, and perichromatin granules within the nuclei of the lymphocytes and the reticular cells were observed frequently. From the above results, it was concluded that DNA syntheses were more active in the cells of the marginal zone than in the cells of the white pulp or the red pulp. And repeated treatment with BCG could activate the DNA syntheses of splenic cells and increase the number of the macrophages and the plasma cells in the white pulp.

Effects of Antitumor Agents on Ultrastructure of the Splenic White Pulp of the Mouse Implanted with Ehrlich Carcinoma Cells / 대한해부학회지

This experiment was performed to study the morphological responses of the splenic white pulp, the lymphatic tissue of the spleen, of Ehrlich carcinoma cell-implanted mice to three different anticancer drugs (5-fluorouracil, mitomycin C and AG60). Healthy adult ICR mice weighing 20 g each were divided into normal and experimental groups. In the experimental groups, each of mice was inoculated with 1X10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day, 0.2 ml of saline solution, 5-fluorouracil (30 mg/kg), mitomycin C (400 microgram/kg) or AG60 (30 mg/kg, Taerim Pharm. Co., Seoul, Korea) were injected subcutaneously every other day, and animals were sacrificed at 14th day following the f irst injection. Pieces of the tissues were taken from the spleen, and prefixed with phosphate buffered 2.5% paraformaldehyde-1.5% glutaraldehyde solution (pH 7.3) followed by post-fixation with phosphate buffered 1% osmium tetroxide solution (pH 7.3). Fixed tissue blocks were dehydrated, and embedded in araldite mixture. Ultrathin sections stained with uranyl acetate and lead citrate were observed with a JEM 100CX-II electron microscope. In the experimental control group (carcinoma cell-inoculated mouse), splenic white pulp did not show pronounced morphological alterations, but myelin figures were frequently observed in the cytoplasm of some lymphocytes and reticular cells than those of normal control mice. In the AG60 treated group, splenic white pulp did not show specific morphological defect, but nuclear bodies and severe invaginations of the nuclear envelope of the lymphocytes and reticular cells were observed occasionally. In the mitomycin C treated group, myelin figures, severe invaginations of the nuclear envelope, nuclear protrusions, nuclear bodies and interchromatin granules were frequently observed in the lymphocytes and reticular cells of the white pulp. In the 5-f luorouracil treated group, myelin f igures, severe invaginations of the nuclear envelope, nuclear protrusions, nuclear bodies and interchromatin granules were observed more frequently in the lymphocytes and reticular cells of the white pulp, as compared with those of mitomycin C treated mice. From the above results, 5-f luorouracil or mitomycin C may suppress the splenic immune function of cancerinoculated mice, since they suppress the process of differentiation and maturation of splenic lymphocyte and reticular cells, and 5-fluorouracil was more harmful on the spleen than mitomycin C. Whereas AG60 does not affect remarkably the process of differentiation and maturation of lymphocytes and reticular cells in the splenic white pulp.

Effects of Lower Dose-Injections of Acriflavine-Guanosine Compound (AG60) on the DNA Synthesis and Ultrastructure of the Ehrlich Carcinoma Cells Inoculated to the Mouse / 대한해부학회지

AG60, a recently introduced anti-cancer compound, was reported to show highly effective anti-cancer activities, when injected with doses from 30 mg/kg to 5 mg/kg.The purpose of this study was to know the lower effective doses of AG60, and to give the informations for preparing more advanced therapeutic tools for anti-cancer war. Ehrlich cancer cells were inoculated in the subcutaneous tissue of inguinal region of ICR mice, and saline (treated control groups) or AG60 (experimental groups) were injected daily. Animals of experimental groups were injected subcutaneously with doses of 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, or 2.0 mg/kg body weight, according to their subgroups. Five mice from each subgroup were sacrificed on 1 week, 2 weeks, 3 weeks following the first injection. Seventy minutes before sacrifice, each mouse was injected with 0.7 microCi/g body weight of 3H-thymidine (Amersham Lab.) through tail vein. After sacrifice, cancer masses were fixed in 10% formalin solution for autoradiography and light microscopy, and in 2.5% glutaraldehyde-1.5% paraformaldehyde solution, followed by post-fixation in 2% osmium tetroxide solution, for electron microscopy. The observed results were as follows: Autoradiographic observations show, 1. Labelled cancer cell indices of the experimental groups received AG60 were decreased around to 80% (0.2 mg/kg), to 74% (0.5 mg/kg), to 75~60% (1.0 mg/kg), and to 70~50% (2.0 mg/kg), as compared with those of the controls. 2. The contents of silver grains were dramatically decreased nearly to 35% (0.2 mg/kg), to 20% (0.5 mg/kg), to 21~16% (1.0 mg/kg), and to 20~15% (2.0 mg/kg), as compared with those of the controls. 3. Total granular content in 100 cancer cells on the third week of the experiment decreased nearly to 30% (0.2 mg/kg), to 15% (0.5 mg/kg), to 10% (1.0 mg/kg), and to 8% (2.0 mg/kg), as compared with those of the controls. Histological observations show, 1. AG60 induces large amount of apoptosis on Ehrlich cancer cells. 2. Following the treatment with AG60, multinuclear cells or giant cells were increased in number. Comparing by autoradiography and histology, multinuclear or giant cells were interpreted as those cells supplied by poor amounts of thymidine, or almost no new DNA content. Electronmicroscopic readings show, 1. AG60 induces numerous macroclefts and microclefts within the nuclei of Ehrlich cancer cells. 2. AG60 induces numerous apoptosis among Ehrlich cancer cells. 3. Apoptotic bodies are phagocytosed by adjacent cancer cells or by macrophages. From the above results, AG60 is expected to be a very successful anti-cancer candidate. And it is suggested that combined or cocktail therapy including AG60 may greatly improve the anti-cancer therapy on certain kind of cancer.