The dynamics of morphological and cytoenzymatic changes in peripheral blood lymphocyte lysosomes in patients with chronic lymphocytic leukemia during treatment.

Changes in the number of lysosomes were studied by method of Blum and the activity of acid phosphatase was determined by the method of Barka and Anderson in the peripheral blood lymphocytes of patients with chronic lymphocytic leukemia in relation to the phase of the disease (according to Rai) and the method of treatment. Treatment with powerful cytostatic agents (cyclophosphamide with vinblastine) significantly changed the fluorescent and cytochemical findings, with a different pattern of changes in cases treated with success (remission) and in cases with unsuccessful treatment (death). The morphological evaluation of lysosomes in the lymphocytes can be used as a simple criterion of success in the treatment, and prognosis in this disease.

Morphological and cytochemical changes of peripheral blood lymphocyte lysosomes in chronic lymphocytic leukemia.

Morphological changes of lysosomes (according to Blum) and the activity of acid phosphatase (by the method of Barka and Andersen) were investigated in peripheral blood lymphocytes in patients with chronic lymphocytic leukemia non-treated or treated with Leukeran. The division into groups was carried out according to Rai. Pathological lymphocytes in relation to lymphocytes of healthy subjects showed a decreased number of lysosomes and an increased number of cells with diffuse type of reaction to acid phosphatase. A relation was found between the intensity of morphological changes of lysosomes in pathological lymphocytes and the clinical state of the patients. Morphological evaluation of lysosomes in lymphocytes is a good diagnostic criterion of treatment effectiveness providing prognostic cues.

Morphology of lysosomes in different phases of growth of lymphoblasts of L5178Y murine leukemia.

Parallel studies were carried out on the morphology of lysosomes, activity of DNA synthesis measured by the rate of 3 H-thymidine incorporation, and proliferative activity on the basis of values of the mitotic index of leukemia L5178Y lymphoblasts under varying growth conditions. Morphology of lysosomes was studied by the method of intravital staining with euchrysine. Observations were made on cells growing from the logarithmic phase to saturation density and synchronized by double blocking of DNA synthesis by hydroxyurea. The results showed some variation of the morphology of lysosomes related to growth phase of the cells.

Mechanism of unbalanced growth-induced cell damage. I. A probable role for hydrolytic enzymes synthesis.

This study examines the relationship between cell progress into the state of unbalanced growth, hydrolytic enzyme activities and cell survival during the exposure of L5178Y cells to hydroxyurea (HU), excess thymidine (dThR), hydroxyurea with excess of four deoxyribonucleosides (dNR) or excess dTHR with deoxycytidine (dCR). Cell progress into the state of unbalanced growth was measured as cell size, protein/DNA ratio and protein content per cell. Activities of two lysosomal (acid phosphatase, beta-N-acetylglucosaminidase) and one cytoplasmic non-lysosomal (LDH) enzymes were determined. It has been found that in cells arrested by HU or excess dThR, a progressive cell volume increase with protein/DNA imbalance is correlated with a progressive increase in lysosomal and non-lysosomal hydrolase activities in the cells and in the medium and with a marked lethal effect. Cell volume increase, enhancement of enzyme activities and cell killing could be prevented in HU-arrested cells by concomitant addition of excess dNR (deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine) leading to equal inhibition of DNA and protein synthesis. Control-like values of all parameters were achieved also in cells in which the dThR-inhibiting effect was reversed by dCR addition. It is suggested that a common pathway in the mode of action of the chemotherapeutic agents inducing cell killing through the state of unbalanced growth can be the over-production, abnormal accumulation and progressive leakage of numerous hydrolytic enzymes through the cell membranes, leading in consequence to 'lytic' cell death.

Voltammetric determination of traces of Cr(VI) in the flow system in the presence of bipyridyne.

The voltammetric method of Cr(VI) determinations in the flow system, based on the combination of selective accumulation of the product of Cr(VI) reduction on HMDE and a very sensitive method of chromium determination in the presence of bipyridine [Z. Gao K.S. Siow, Electroanalysis 8 (1996) 602] is proposed. The calibration graphs were linear from 3x10(-9) to 3x10(-8) and from 5x10(-10) to 5x10(-9) mol l(-1) for accumulation times 120 and 600 s, respectively. The relative standard deviation (R.S.D.) was 6.5% (n=5) for Cr(VI) concentration 1x10(-8) mol l(-1) and the accumulation time 120 s. The influence of foreign ions commonly present in water samples is presented. Validation of the method was made by comparison of the results of analyses of tap water by another electrochemical method and by recovery test for river and mineral water.

Alteration in lysosome supravital staining as a marker of hydroxyurea-induced cytotoxicity and its modification by radical scavengers in L5178Y cells in culture.

Supravital staining of lysosomal membranes with euchrysine was employed to study hydroxyurea (HU)-induced side toxic effects in L5178Y lymphoblasts in culture. Exposure of cells to 0.1-1.0-10.0 mM HU-induced progressive increase in the proportion of cells without detectable lysosomal fluorescence. This effect preceded the occurrence of non-viable cells, determined by trypan-blue exclusion test. Addition of alpha-tocopherol, acetylsalicylic acid, sodium benzoate or ascorbic acid to the culture medium afforded a concentration-dependent modification of lysosomal response to HU treatment. It is suggested that the fluorescence technique of supravital lysosome staining can be a useful test in studies on the side toxic effects of free radical-forming drugs and their amelioration by radical scavengers.

Hydroxyurea-induced toxic side-effects in animals and an attempt at reducing them with vitamins E and C.

Using the changes in several blood parameters and in the histological picture of the liver as markers of toxicity, the effect of hydroxyurea (HU) in healthy rabbits or mice was examined during two weeks after a single administration of this drug. In rabbits a transient significant decrease in blood erythrocyte count with a gradual increase in their osmotic resistance, a suppression of granulocyte phagocytic capacity, and an elevation of acid phosphatase activity in the serum were found. An increase in the proportion of lymphocytes without detectable lysosomes as tested by supravital staining also appeared. No significant difference was observed in the white cell count and lipoperoxide levels after HU administration. Histological picture of the liver, excised from HU-treated mice, indicated a marked hepatotoxicity of the drug. Some of the toxic effects were reduced in animals supplemented with vitamins E and C.

Erythro-toxicity of dichlorvos and possibility of its modification in vitro.

The purpose of this study was to investigate the in vitro effects of dichlorvos (organophosphorous pesticide) on the biological value of erythrocytes; an in vitro test of erythrocyte protection against DDVP effects was also included. A significant decrease of glutathione level with concomitant increase of methemoglobin and denatured hemoglobin levels, as well as a rise of Heinz body number were observed after 24 h following the treatment with dichlorvos (100 microM). Preincubation of erythrocytes with glutathione prevented the toxic effects of the pesticide with the maintenance of both glutathione and methemoglobin at a control level.

[Protection of human peripheral blood leukocytes with vitamin E and C from toxic effects of fenarimol in vitro]. / Oslanianie leukocytów ludzkiej krwi obwodowej za pomoca witamin E i C przed skutkami toksycznego dzialania fenarimolu in vitro.

The work was undertaken in a trial to protect leukocytes against the toxic effect of fenarimol with vitamin E and C. The experiment was carried out in vitro. These vitamins were chosen since they are the components of the physiological antioxidative mechanism in the cell. The protective effect of the vitamins varied depending on the type of the cell and the studied structure as well as on the dose of the antioxidant used. Improvement of the phagocytic activity of the granulocytes was observed after preincubation with vitamin E and using vitamin C it was possible to achieve control values of the phagocytosis index and per cent of non-phagocytizing granulocytes. Both these vitamins in a satisfactory degree improved the morphological and quantitative pattern of lysosomes in cultured lymphocytes treated with fenarimol. The use of vitamin E in doses of 15 micrograms/ml and vitamin C - 0.5 microgram/ml, made possible achieving of complete normalization of the nucleologram in the lymphocytes. The observed effective protection of the leukocytes against the effects of fenarimol by means of antioxidants suggests that these cells are damaged by the mechanism of lipid peroxidation.

[Cytotoxic effect of fenarimol on peripheral blood cells and organs]. / Cytotoksyczne dzialanie fenarimolu na komórki krwi obwodowej i narzady.

The effect of fenarimol in blood cells and changes in liver and kidneys in rats were investigated. Fenarimol applied in doses 25 mg/ml intragastrically during 5 days, at 24h after the last doses caused functional and structural changes of blood cells and organs. The decreases of osmotic fragility of erythrocytes and phagocytic activity of neutrophils, and the increase of the percentage of lymphocytes without detectable lysosomes accompanied by the minor decrease of the number of blood cells was observed in the peripheral blood. The indistinctness of the structure of the hepatic lobules and of liver laminae and the vacuolized hepatic cells with pycnotic nuclei were visible in the liver. The abnormally enlarged cortical Malphighian++ glomeruli in kidney were found. The observed changes indicate the cytotoxic effect of fenarimol in strongly intoxicated rats. The impaired phagocytic activity of neutrophils may cause the growth of susceptibility to bacterial infection.

[Lysosomes of blast cells in various cytochemical types of acute leukemia]. / Obraz lizosomow komórek macierzystych w róznych typach cytochemicznych ostrych bialaczek

In 25 cases cytochemical types of acute leukemia were determined using the classification of Loeffler. Three cytochemical methods used were: p.a.S.-glycogen content, pox-peroxidase activity, and esterase activity determinatione. At the same time selective identification of lysosomes in perypheral blood blast cells was done using vital euchrisine staining and fluorescence microscopy. A correlation was observed between the cytochemical types of acute leukaemia and the lysosomal pattern. The fluorescence method of the identification of lysosomes is suggested for diagnosis of acute leukaemia.

Protective action of vitamin E on the subcellular structures of lymphocytes intoxicated with pesticides in vitro.

Vitamin E was used as a protection for human lymphocytes in vitro against the toxic effects of dichlorvos, an organic phosphate pesticide. As the markers of the toxic changes in the lymphocytes lysosomes were chosen as a membrane structure, and nucleoli as a structure connected with protein synthesis. The obtained results were compared with the effects obtained previously in analogous studies after intoxication of lymphocytes with lindan and fenarimol. The experiment showed that vitamin E protected the lysosomal membranes more effectively than the nucleolar structure in these cells.

Protective effects of vitamins E and C on erythrocytes in blood preserved in ACD solution and stored at 4 degrees C.

Vitamins E and C were used for the enrichment of the fluid for blood preservation. Whole blood with added ACD solution and vitamins E and C was stored at 4 degrees C for 21 days. The performed determinations included: haematocrit, osmotic fragility of the erythrocytes, ATP and 2,3-DPG content in the erythrocytes, and echinocyte count. A protective effect of these vitamins on erythrocytes was manifested by the decreased osmotic fragility, the steep fall of 2,3-DPG compared with the blood preserved in the vitamin-free ACD solution.

Hydroxyurea has the capacity to induce damage to human erythrocytes which can be modified by radical scavengers.

The treatment of human erythrocytes with hydroxyurea [HU] results in the azide-dependent changes in osmotic fragility and in increased methemoglobin formation. Similar changes were induced by H2O2 treatment. However when H2O2 in the presence of azide stimulated malondialdehyde production, in the HU-treated cells no malondialdehyde was detectable. When subjected to an oxidant stress [sodium ascorbate] HU-treated erythrocytes were more fragile and revealed changes in the absorption spectrum of the TBA-reactive material in comparison with the cells treated with ascorbate alone. Partial protection by radical scavengers against certain HU-induced changes can be achieved. The results indicate that HU can damage erythrocytes and suggest the radical origin of these effects.

Vitamin E protects human leucocytes against toxic effects of lindan in vitro.

Vitamin E (15 micrograms/ml and 60 micrograms/ml) was used for protection of human leucocytes against toxic effects of lindan (150 microM and 200 microM) in vitro. The protective effect of vitamin E manifested itself as maintenance of the phagocytic activity of granulocytes, the value of nucleologram in lymphocytes, and the proportion of euchrysin-negative lymphocytes near the values in controls.

Late toxic effects of long-term exposure to lindane in peripheral blood cells in rabbits. I. Function impairment and structural disturbances in leucocytes.

Following lindane intoxication in daily doses of 0.1 LD50 during one month (total dose 2.0 LD50) functional changes were noted in granulocytes and structural changes in lymphocytes in peripheral blood of rabbits during 4 months after completion of lindane administration. A significant decrease of the phagocytic activity with a rise in the number of non-phagocytizing granulocytes, quantitative and qualitative changes of nucleoli and lysosomes were found in lymphocytes. The latest effect of lindane is regarded to be a raised number of non-phagocytizing granulocytes 4 months after withdrawal of the pesticide.

Late toxic effects of long-term exposure to lindane in peripheral blood cells in rabbits. II. Erythrocyte changes.

After lindane administration in daily doses of 0.1 LD50 for one month (total dose 2.0 LD50) changes in the erythrocytes were found persisting for up to 4 months after withdrawal of the pesticide. A significant increase in the number of echinocytes, a greater per cent of reticulocytes, significantly raised level of denatured haemoglobin, and an increased number of erythrocytes of higher sensitivity to oxidating factors in relation to control group was observed.

The effect of gamma-hexachlorocyclohexane (lindane) on blood cells, kidney and liver tissues in rabbits.

The effect of intragastric administration of gamma-hexachlorocyclohexane (lindane) on peripheral blood cells, kidney and liver was studied in rabbits. White blood cells were found to be most affected by lindane, as was shown by the reduced phagocytic activity of neutrophils and the increased number of lymphocytes with inactive nucleoli. The changes in the characteristics of erythrocyte cell membranes depended on the length of time lindane was administered and on its withdrawal. Histological examination of the liver and kidney showed focal degeneration of tissue and cell structure.