M. Topashka-Ancheva

Alkylphosphocholines: Effects on human leukemic cell lines and normal bone marrow cells

The anti‐leukemic activity of a series of alkylphosphocholines (APCs) was studied against a panel of human leukemic cell lines (HL‐60, K‐562, Reh, MOLT‐4, Jurkat, Ramos and Raji). Cytotoxic efficacy was measured by the MTT cell survival assay. All cell lines were found to be sensitive, except the multipotential CML‐derived K‐562 cell line. Flow cytometry of HL‐60 cells showed a significant decrease of cells in S phase and the formation of a sub‐G1 fraction. DNA fragmentation typical for programmed cell death was detected by DNA gel electrophoresis in these cells but not in any of the other leukemic lines. At concentrations below the cytotoxic range, mitogenic effects were seen in HL‐60 cells after 14‐hr exposure. Colony formation by K‐562 cells revealed an augmented clonogenicity after exposure to APC with a short alkyl chain. In contrast, cells of lymphoid origin did not undergo DNA fragmentation or show mitogenic stimulation after exposure to APC. Normal bone marrow cells were also investigated for mitogenic and genotoxic effects. No decrease was found in the number of hematopoietic progenitors in long‐term bone marrow cell cultures after exposure to APC. On the contrary, a significant increase was found after short exposure. Dodecylphosphocholine, hexadecylphosphocholine (HPC) and (octadecyl‐[2‐(N‐methylpiperidino)‐ethyl]‐phosphate exhibited a mild clastogenicity at equimolar high doses on murine bone marrow cells in vivo,which is unusual for the majority of classical DNA‐interacting anti‐cancer drugs. In conclusion, APCs are agents with a broad spectrum of in vitro anti‐leukemic effects, which lack hematological toxicity. Int. J. Cancer,77:778–786, 1998.

Cytotoxic efficacy of bendamustine in human leukemia and breast cancer cell lines.

Abstract.Purpose: The purpose of this study was to characterise bendamustines cytotoxic and apoptotic activity in a panel of leukemia and breast cancer cell lines in comparison to its clastogenicity in murine bone marrow. Methods: The cytotoxic effect of bendamustine was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-dye reduction assay. Induction of apoptosis was evidenced by DNA gel electrophoresis, nuclear staining, Western blot poly-(adenosine diphosphate-ribose) polymerase (PARP) cleavage, and flow cytometry. As a measure of hematological toxicity, the formation of chromosomal aberrations was investigated in bone marrow cells isolated from mice treated with low non-toxic doses of bendamustine and lomustine. Results: Bendamustine was preferably active against leukemic cells of lymphoid origin and was found to induce apoptosis in SKW-3 and BV-173 cells as shown by oligonucleosomal DNA and nuclear fragmentation, PARP cleavage, and formation of a sub-G1 fraction. Myeloid and breast carcinoma cell lines were resistant towards bendamustine with the exception of HL-60 cells which exhibit an intermediate sensitivity. Bendamustine was found to have a very low clastogenic effect as compared with equimolar doses of lomustine. Conclusion: Taken together, the mode of action of bendamustine includes induction of apoptosis. The specific spectrum of activity and the unexpectedly low clastogenicity support the hypothesis that bendamustine in not a typical alkylating agent but exerts an additional mode of action, possibly as a purine antimetabolite.

Genotoxic effect of substituted phenoxyacetic acids.

Abstract. The potential toxic and mutagenic action of 2,4-dichlorophenoxyacetic acid has been studied in different test systems, and the obtained results range from increased chromosomal damage to no effect at all. We reexamined the effect of this herbicide by simultaneous using three tests based on yeast, transformed hematopoietic, and mouse bone marrow cells. The results obtained demonstrated that 2,4-dichlorophenoxyacetic acid has cytotoxic and mutagenic effects. The positive response of yeast and transformed hematopoietic cells was verified in kinetics and dose–response experiments. The analysis of metaphase chromosomes indicated a statistically proved induction of breaks, deletions, and exchanges after the intraperitoneal administration of 2,4-dichlorophenoxyacetic acid in mice. The study of phenoxyacetic acid and its differently chlorinated derivatives showed that cytotoxicity and mutagenicity are induced by chlorine atoms at position 2 and/or 4 in the benzene ring. The mutagenic effect was abolished by introduction of a third chlorine atom at position 5. Thus 2,4,5-trichlorophenoxyacetic acid was found to have very weak, if any mutagenic effect; however, the herbicide preserved its toxic effect.

Antineoplastic and Anticlastogenic Properties of Curcumin

Abstract:  Curcumin is the pigment of turmeric and has been reported as a signal transduction modulator and inhibitor of transcription factors, for example, NF‐κB. In our article we found a concentration‐dependent cytotoxic activity of curcumin in a panel of eight leukemic cell lines (SKW‐3, CEM, U‐937, HL‐60, HL‐60/Dox, K‐562, LAMA‐84, and AR‐230). Additive to synergistic interactions was recorded for combinations with bendamustine and idarubicine in SKW‐3 and LAMA‐84 cells. Noteworthy, in multiple myeloma cells (RPMI‐8226 and U‐266) a potentiation of the efficacy of bendamustine by curcumin application was found. Moreover, curcumin increased the bendamustine cytotoxicity in cultures of cells isolated from the bone marrow of a patient with non‐Hodgkins lymphoma (NHL). The increased bendamustine efficacy could be explained by NF‐κB inhibition, because this factor is activated in many cancers, especially leukemia and multiple myeloma. Curcumin is characterized by low toxicity and was described to have a chemoprotective activity. Therefore, the level of reduced glutathione (GSH) was measured and a concentration‐dependent increase of GSH levels was recorded in AR‐230 and SKW‐3 cells (concentration range 5–25 μM). Experiments with mice showed significant protection against cisplatin‐induced chromosomal aberrations (clastogenic effect) and inhibition of mitoses in bone marrow cells. Curcumin alone caused reduction of the mitotic index. In combination with cisplatin, however, this parameter was increased when compared to cisplatin alone. Our data indicate that curcumin has pleiotropic effects on signal transduction by inhibiting transcription thus exerting antitumor activity. In addition, curcumin has protective and anticlastogenic activity by enhancing the scavenging of free radicals.

A comparative analysis of the heavy metal loading of small mammals in different regions of Bulgaria II: chromosomal aberrations and blood pathology.

Heavy metal content was monitored in small mammals inhabiting mountain ecosystems and two industrial polluted regions in Bulgaria. Rodents (Microtus arvalis, M. rossiaemeridionalis, Clethrionomys glareolus, Pitymys subterraneus, Chionomys nivalis, Apodemus flavicollis, A. sylvaticus, and Mus macedonicus) were used as zoomonitors. Pathological changes in chromosome status, hematological indices, and blood cell morphology were analyzed in the context of heavy metal bioaccumulation. Significant correlations were obtained between the heavy metal load of zoomonitors and the frequency of chromosomal aberrations and pathological changes in erythrocytes (mainly micronuclei and basophilic granulations). It is suggested that mercury is a strong damaging factor for chromosomes and red blood cell apparatus.

Synthesis, cytotoxicity and clastogenicity of novel α-aminophosphonic acids

Summary.α-Ethyl-N-(phosphonomethyl) glycine is synthesized and characterized by NMR and FAB spectroscopy. The cytotoxicity, clastogenic and antiproliferative effect of 3-ethyl-2-hydroxyl-2-oxo-1,4,2-oxazaphosphorinane, sodium salt of 3-ethyl-2-hydroxyl-2-oxo-1,4,2-oxazaphosphorinane, α-ethyl-α-N-(hydroxyethylamino) methylphosphonic acid, α-ethyl-N-(phosphonomethyl) glycine, α-ethyl-N-(phosphonomethyl) glycine isopropylammonium salt, glyphosate isopropylammonium salt are tested.

Synthesis, antiproliferative activity and genotoxicity of novel anthracene-containing aminophosphonates and a new anthracene-derived Schiff base.

A new Schiff base, 9-anthrylidene-furfurylamine and three novel anthracene-containing α-aminophosphonates, [N-methyl(dimethoxyphosphonyl)-1-(9-anthryl)]-p-toluidine, [N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]-p-toluidine and [N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]furfurylamine were synthesized. The compounds have been characterized by elemental analysis, TLC, IR, NMR and fluorescent spectra. The aminophosphonates and their synthetic precursors were tested for in vitro antitumor activity on a panel of seven human epithelial cancer cell lines. Safety testing was performed both in vitro (3T3 NRU test) and in vivo on ICR mice for genotoxicity and antiproliferative activity. 9-Anthrylidene-furfurylamine and [N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]furfurylamine were most potent cytotoxic agents towards colon carcinoma cell line HT-29. The latter compound exhibited also antiproliferative activity to HBL-100, MDA-MB-231 and 647-V cells. The aminophosphonate [N-methyl(dimethoxyphosphonyl)-1-(9-anthryl)]-p-toluidine and its synthetic precursor 9-anthrylidene-p-toluidine were found to be cytotoxic to HBL-100 and HT-29 tumor cell lines, respectively. Moderate genotoxic and antiproliferative activity in vivo and low toxicity to Balb/c 3T3 (clone 31) mouse embryo cells were observed for all tested compounds. The subcellular distribution of two tested compounds in a tumor cell culture system was also studied.

Bioaccumulation and damaging action of polymetal industrial dust on laboratory mice Mus musculus alba: I. Analysis of Zn, Cu, Pb, and Cd disposition and mathematical model for Zn and Cd bioaccumulations☆

The concentrations of Zn, Cu, Pb, and Cd in the liver, kidneys, spleen, bones, and carcass of laboratory mice BALB/cy were observed in toxicological experiments. Polymetal industrial dust containing these metals was given to experimental animals at 1% concentration mixed with conventional animal food. Samples for analyses were taken on Days 15, 40, 60, 90, and 120 posttreatment. The experimental data clearly support the established antagonistic interactions among cadmium, zinc, copper, and lead. A mathematical model was proposed to study the main tendencies of heavy metal bioaccumulation under conditions of metal interaction and excessive exposure. The experimental results were assessed on the basis of the model. A rate constant of renal excretion greater than that of hepatic excretion was obtained, which agrees with the observed inversion of cadmium kidney/liver ratio in the conditions of very high exposure.

Modified Natural Clinoptilolite Detoxifies Small Mammal’s Organism Loaded with Lead I. Lead Disposition and Kinetic Model for Lead Bioaccumulation

Zeolites, especially clinoptilolites, have wide application in removing heavy metals from different solutions and wastewater. The detoxification capacity of the clinoptilolite sorbent KLS–10-MA, a modified natural Bulgarian zeolite, applied as a food supplement in conditions of an ecotoxicological experiment with conventional food and lead was demonstrated for the first time. Laboratory mice, inbred imprinting control region strain, were used in a 90-day ecotoxicological experiment. Animals were divided into four experimental groups. Lead bioaccumulations in exposed and non-supplemented/supplemented with KLS–10-MA animals were compared. As additional control, healthy animals non-exposed to Pb were fed with conventional forage mixed with 12.5% KLS–10-MA. The dietary inclusion of the sorbent reduced Pb concentrations in exposed and supplemented mice by 84%, 89%, 91%, 77%, and 88% in carcass, liver, kidneys, bones, and feces, respectively. A mathematical model was proposed to outline the common trends of bone Pb bioaccumulation in exposed and non-supplemented/supplemented animals. Characteristic parameters of the kinetics of Pb concentrations were determined. Based on the model, the coefficient of absorption of Pb by gastrointestinal mucosa in the supplemented mice was found—η = 3.53% (versus η = 15% in non-supplemented ones). The present study clearly indicates that there is a realistic perspective to create a new drug based on modified natural clinoptilolites in cases of chronic heavy metal intoxication, without negatively affecting the environment.

A comparative analysis of the heavy metal loading of small mammals in different regions of Bulgaria: I: monitoring points and bioaccumulation features

Data on liver and body copper, zinc, lead, and cadmium content of small mammals (rodents and insectivorous) were collected and analyzed. Data comparisons were performed in two aspects: (1) points and years of monitoring; (2) monitor species bioaccumulations. Specific bioaccumulation features were observed in some of the monitor species. A method for comparative evaluation of heavy metal loads in the different species is proposed using data for liver and body contamination. The loads of Clethrionomys glareolus and Apodemus flavicollis were compared, and the data are in agreement with data from other authors in Central Europe. A correlation between heavy metal content in the food and liver of snow vole was established. The data demonstrate that two of the regions investigated in Rila Mountain National Park could be assumed to be background locations. Some possible reasons for the heavy metal contamination of the low-altitude region in Rila are discussed. Not very significant pollution was observed around industrial facilities. Correlations between heavy metal levels in zoomonitors and meteorological factors were established.