Ultraviolet Radiation Effects on Ehrlich Ascites Tumor Cells : Observations Using a Flying Spot Ultraviolet Microscope

A flying spot ultraviolet microscope, employing a fast scan and pulsed operation of the raster, has been used to induce radiation damage in ascites tumor slide cultures, and to study by time-lapse cinematography the progressive stages of cell damage. The cells observed came from a strain (EF₇) of the Ehrlich ascites carcinoma. Irradiated cells were found to show a characteristic syndrome of damage, involving blebbing at the cell surface, while control cells in the adjacent areas of the preparation remained unchanged. The end of the blebbing period is marked by swelling of the cells, and the time taken for this phenomenon to occur was used as a measure of the severity of the damage. It was found that the time required for swelling is dependent on the size of the dose employed, as well as on the sensitivity of the cells. This latter sensitivity was found to decline as the physiological age of the tumor increased. If ultraviolet illumination below 255 mµ is excluded, no symptoms of damage occur, even when very large doses are used. These observations are discussed in relation to the nature of the system in the cell which is affected.     

Cell surface changes in a cell line derived from a human pleural effusion

The phenomenon of blebbing has been recorded in cells obtained from a pleural effusion of a patient with a small cell lung carcinoma. The blebbing manifested itself as a sudden ballooning of the cell membrane which then moved around the perimeter of the cell as a sausage-shaped swelling. This was followed by its rapid collapse. The rapidity of the process allowed real time video recordings, not time lapse, and still frames from the recording were photographed. Cells were also viewed using a scanning electron microscope and more detail of the blebs was revealed. Possible functions of cell blebbing are discussed.     

Effect of electromagnetic microwave radiation on the growth of Ehrlich ascites carcinoma

Daily exposure of mouse recipients of Ehrlich ascites carcinoma to electromagnetic radiation of the microwave range leads to a change in the dynamics of tumor growth by decreasing the total number of cells. The number of tumor cells with blebbing morphological signs after microwave radiation increases gradually with tumor growth. The maximum content of tumor cells in the state of blebbing is observed during active proliferation in tumor-recipient mice of the control group (without irradiation).     

Assessment of the cyto- and genotoxic effects of a nanoferromagnetic and a static magnetic field in vivo

The cyto- and genotoxic effects of ferromagnetic nanoparticles (FNs), a static magnetic field (SMF), or their combination were comparatively studied in Ehrlich ascites carcinoma (EAC) cells, bone marrow (BM) erythroid series cells, and peripheral blood lymphocytes at definite time intervals, using the MN test and the DNA-comet assay. The MN test and the DNA-comet assay were used as markers of genotoxic exposure. It has been shown that the limit value of insignificantly expressed changes in the EAC cell architectonics (due to FNs) is an FN concentration of 3 mg/kg. The FN concentration increases in tumor cells, because the expressed blebbing in the cytoplasmic membrane increases the amount of micronuclei and the percentage of DNA in the comet tail in EAC cells, BM erythroid series cells, and peripheral blood lymphocytes. It has also been demonstrated that an SMF alone, as an independent factor, does not produce cytogenotoxic effects on the studied cells. When SMF and FN were used in combination, we could observe the phenomenon of SMF induction by FNs, which manifested itself as an increased total effect of these factors on cells. The MN-test and the DNA-comet assay can be used for assessing the genomic stability of both tumor cells and somatic nonmalignized cells when testing the genotoxic effect of nanomaterials used in the design of vector systems. As was established, the DNA-comet method demonstrated a higher sensitivity in the assessment of the genotoxicity produced by the studied factors.     

Shape and Volume Changes in Frog Erythrocytes Following Ultraviolet Radiation

Frog erythrocytes in Ringer's solution were exposed to ultraviolet radiation and then followed in camera lucida drawings for changes in shape and dimension. Cell thickness was found to increase while cell width remained constant throughout the period prior to hemolysis. The cell shortened and bulged at the ends during the middle third of the prolytic period while a region around the cell center remained constricted. When this constricted region gave way, the cell became spherical and hemolyzed. Cell volume as calculated from the cell's dimensions increased linearly with time throughout the prolytic period to hemolysis then dropped rapidly to a constant value somewhat higher than the original cell volume. These changes in shape and volume are consistent with a colloid osmotic type of hemolysis but with other factors acting to limit the rate of swelling and the forms assumed during the swelling process. The relationship between the time of hemolysis and the cell surface area exposed to the ultraviolet is discussed as it applies to the site of ultraviolet damage.     

Variation of cell kinetic parameters in relation to the growth rate of the Ehrlich ascites tumor.

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

VARIATION OF CELL KINETIC PARAMETERS IN RELATION TO THE GROWTH RATE OF THE EHRLICH ASCITES TUMOR

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

Aeromonas hydrophila exotoxin induces cytoplasmic vacuolation and cell death in VERO cells

Many organisms are able to cause cell vacuolation, but it is unclear if this can be considered a step of apoptosis or necrosis, or a distinct form of cell death. In this study VERO cells were used to evaluate the relationship between vacuolation and cell death pattern caused by exotoxins produced by environmental strains of A. hydrophila. Cell damage has been evaluated morphologically as well as biochemically. Cytotoxic and vacuolating titres were strictly correlated and the vacuolation has to be considered an early indicator of cytotoxicity that causes cell apoptosis or necrosis in relation to the dose. Signs of apoptosis (chromatin condensation and blebbing) were observed at low concentration and TGase activity, referable to apoptosis induction, confirms morphological observations. In fact, putrescine incorporation was related both to cytotoxin concentration and time of incubation. Moreover, the observed doubling cells with necrotic features permit us to suppose that cell sensitivity and death pattern could change during the different phases of cellular cycle.     

Cell Swelling, Blebbing, and Death Are Dependent on ATP Depletion and Independent of Calcium During Chemical Hypoxia in a Glial Cell Line (ROC-1)

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligodendroglial-glioma hybrid cells (ROC-1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to less than 5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehydrogenase release) which began at approximately 20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K(+)-ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to approximately 10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at approximately 55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracellular calcium ([Ca2+]i) was measured using fura-2. In calcium-containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 +/- 17 nM) followed by a low steady-state level preceding plasma membrane disruption. Following deenergization in calcium-free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.     

Regulation of cell volume by glycosaminoglycans

Cell volume is regulated by a delicate balance between ion distribution across the plasma membrane and the osmotic properties of intra‐ and extracellular components. Using a fluorescent calcein indicator, we analysed the effects of glycosaminoglycans on the cell volume of hyaluronan producing fibroblasts and hyaluronan deficient HEK cells over a time period of 30 h. Exogenous glycosaminoglycans induced cell blebbing after 2 min and swelling of fibroblasts to about 110% of untreated cell volume at low concentrations which decreased at higher concentrations. HEK cells did not show cell blebbing and responded by shrinking to 65% of untreated cell volume. Heparin induced swelling of both fibroblasts and HEK cells. Hyaluronidase treatment or inhibition of hyaluronan export led to cell shrinkage indicating that the hyaluronan coat maintained fibroblasts in a swollen state. These observations were explained by the combined action of the Donnan effect and molecular crowding. J. Cell. Biochem. 113: 340–348, 2012. © 2011 Wiley Periodicals, Inc.     

Lipid peroxidation and cytotoxicity of Ehrlich ascites tumor cells by ferric nitrilotriacetate

Ferric nitrilotriacetate, which causes in vivo organ injury, induced lipid peroxidation and cell death in Ehrlich ascites tumor cells in vitro. The process was inhibited by butylated hydroxyanisole and enhanced by vitamin C and linolenic acid, indicating a close relationship between cytotoxicity and the lipid peroxidizing ability of Fe3+ NTA. The cytotoxicity was suppressed by glucose and a temperature below 20 degrees C. Lipid peroxidation of Fe3+ NTA-treated cells was greater at 0 degree C than at 37 degrees C, contrary to results with Fe3+ NTA-treated plasma membranes of Ehrlich ascites tumor cell. These results suggested that metabolism and membrane fluidity are important factors in the expression of the Fe3+ NTA-induced cytotoxicity. H2O2 showed a lower cytotoxicity than did Fe3+ NTA but a greater lipid peroxidizing ability. H2O2 appeared to damage the cells less, and was quenched rapidly by cellular metabolism unlike Fe3+ NTA. In transferrin-free medium, Ehrlich ascites tumor cell readily incorporated Fe3+ NTA, and iron uptake was greater than NTA-uptake in Fe3+ NTA-treated cells, suggesting that Ehrlich ascites tumor cell incorporated iron from Fe3+NTA and metabolized it into an inert form such as ferritin.     

The effect of polycations on cell membrane stability and transport processes

Summary The interaction of poly-l-lysines of different molecular weights (PL) with Ehrlich ascites tumor cells was studied experimentally with respect to cell surface binding, cell electrophoresis, cytotoxicity and membrane permeability. Although they decrease the net negative charge of Ehrlich ascites cells similarly at low PL concentrations, low molecular weight PL was less cytotoxic and less damaging to the potassium transport mechanism than was high molecular weight PL. At certain PL concentrations, membrane damage was reversible on reincubation in PL-free media. The amount of bound polylysine as determined with fluorescent labeled polylysine was compared by electrophoresis to the amount of polylysine expressed on the electrokinetic surface. The results indicated that only a small fraction of polylysine bound to Ehrlich ascites tumor cells was electrokinetically detectable. The adsorption of polylysine to Ehrlich ascites tumor cells was not describable by the usual adsorption isotherms. It is suggested that the same number of monomeric lysine units of high and low molecular weight PL are adsorbed at the cell electrokinetic surface, but cytotoxicity is dependent on molecular weight. Although the negative charge of human red blood cells could be reversed at low PL concentrations, no such effect could be observed for ELD (a subline of Ehrlich ascites carcinoma) cells even at high PL concentrations. The relationship of PL binding to the stimulation of macromolecular uptake is discussed.     

Patterns in the combined action of irradiation and glucose loading on tumor cells

It was shown that incubation of Ehrlich ascites tumor cells with glucose or in buffer solutions of low pH decreases their viability. The cell survival rate depends on pH values irrespective of the protoxidation method and oxygenation conditions used. At the same time, radiosensitivity of Ehrlich ascites tumor cells is practically invariable with pH being decreased from 7.0 to 5.0. The effects of glucose and radiation are additive in conditions simulating the effect of hyperglycaemia in vivo.     

Association of blebbing with assembly of cytoskeletal proteins in ATP-depleted EL-4 ascites tumour cells.

ATP depletion in EL-4 ascites tumour cells rapidly induced the changes in cell morphology (blebbing), cytoskeletal protein assembly and finally resulted in cell death. After 1 hr of incubation with 2 microM rotenone (inhibitor of respiration) in glucose-free medium, when ATP level was 4% of the initial level, there were increases in triton-insoluble actin and vinculin levels (2.5-fold and 2.8-fold, respectively) and 44% of cells showed blebs; such treatment damaged cells irreversibly. Ca2+ removal did not diminish the effect of ATP depletion on cytoskeleton, blebbing and cell death, although the elevation of free intracellular Ca2+ in rotenone-treated cells was prevented. The role of ATP in maintaining cytoskeleton and cell shape is discussed.     

Lack of evidence of chalone activity in used medium and extract of JB-1 tumor cells in vitro. A flow cytometry study.

In cell-free mouse ascites fluid from the JB-1 ascites tumor in the plateau phase of growth low-molecular chalone substances have been found which reversibly and specifically arrest JB-1 cells in the G1 and G2 phase of the cell cycle. The aim of this study was to investigate whether chalones were involved in the regulation of in vitro growth of JB-1 tumor cells. Used medium and cell extract from confluent, stationary JB-1 cell cultures were investigated for proliferation-inhibitory properties. JB-1 cells from stationary cultures were explanted in test cultures and the traverse of cells through the S phase was investigated by means of flow cytometry (FCM). Inhibition--expressed as a delay of the traverse of cells through the S phase--was not observed when a surplus of used medium, concentrated and fractionated used medium or concentrated and fractionated cell extract from JB-1 cells in vitro was added to test cultures. On the contrary, used medium and concentrated and fractionated used medium stimulated growth. Thus, no involvement of chalones in the growth regulation of JB-1 tumor cells in vitro was detected.     

Effects of trypsin on X-ray-induced cell killing, chromosome abnormalities and kinetics of DNA repair in mammalian cells

When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells. Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines. To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair. Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair. However double-strand break (dsb) repair was unaffected by trypsin. It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair. The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.     

Negatively charged RNase-susceptible molecules on the surface of ascites tumor cells

RNase-susceptible ionogenic groups on the cell surface membranes of two leukemic and two nonleukemic strains of ascites tumor cells were studied by cell electrophoresis, DEAE-Sephadex A-25 column and paper chromatography, and indirect membrane immunofluorescence. RNase treatment of the nonleukemic ascites tumor cells (Ehrlich ascites tumor and Sarcoma 180) produced a significant reduction in their electrophoretic mobilities. When the cells were labeled with [3H]uridine then incubated with RNase, there was a marked increased in the radioactive nucleotides present in the incubation medium as compared to the results of the experiment with RNase-untreated controls. Indirect membrane immunofluorescence studies of nonleukemic ascites tumor cells suggest that the sites that react with anti-RNA antibody are distributed diffusely on their surfaces. RNase treatment of these cells markedly reduced their ability to react with the antibody. It thus appears that RNAs are present on the surface membrane of nonleukemic ascites tumor cells and that RNase digests these RNAs, removing negatively charged nucleotides from their electrophoretic surfaces. This results in a reduction in mobility. In contrast, leukemic ascites cells (L1210 and C1498) incubated with RNase showed no significant change in mobility or in the amount of nucleotides released into the incubation medium. Moreover, no fluorescence was found on the surface of cells examined by indirect membrane immunofluorescence. This suggests that leukemic ascites cells are devoid of RNAs on their surface.     

Pretreatment growth environments alter the sensitivity of tumor cells to cytotoxic agents

The effects of pretreatment growth conditions on the sensitivity of tumor cells to various cytotoxic agents were investigated using murine Ehrlich ascites tumor cells grown in two different environments. The tumor cells adapted to grow in the peritoneal cavity of mice were found to be more sensitive to ionizing radiation, oxygen toxicity, doxorubicin, and bleomycin than tumor cells adapted to grow in vitro. However, there was no difference in their sensitivity to 5-fluorouracil. One obvious difference between these two growth environments is oxygen tension; it is between 2.6 and 5.2% (20-40 mmHg) for the peritoneal cavity and 21% (159 mmHg) for the regular tissue culture. To investigate the role of oxygen tension, tumor cells from the peritoneal cavity were grown in tissue culture having either 21% O2 or 4% O2 in the gas phase. Within 4 d, tumor cells that were exposed to 21% O2, but not to 4% O2, in vitro gradually became as resistant to cytotoxic agents as the tumor cells continuously cultured in vitro under 21% O2. It appears that the adaptation of tumor cells to different environments having different partial pressure of oxygen alters their sensitivity not only to oxygen toxicity but also to other cytotoxic agents that damage or kill cells by generating free radicals.     

Ultrastructural effects of low dosage endocrine disrupter chemicals on neural cells of the chicken embryo model.

Previous research suggests that endocrine disrupters (EDCs) like nonylphenol cause apoptosis (both via the intrinsic and extrinsic pathway) and that ROS generation and Ca (2+) play a fundamental role in the process. We have investigated morphological changes induced by 17beta-estradiol, nonylphenol, 17alpha-ethynylestradiol and diethylstilbestrol on the IN OVO neural chick embryo model by using transmission and scanning electron microscopy (TEM and SEM). We found that estrogenic substances such as nonylphenol, diethylstilbestrol (DES) and 17alpha-ethynylestradiol, as well as 17beta-estradiol cause ultrastructural changes to developing neurons, resulting in damage to the plasma, mitochondrial as well as nuclear membranes. Furthermore, both apoptotic blebbing and necrotic (or oncotic) budding was seen in TEM and SEM micrographs. SEM shows that nonylphenol-exposed neurons have irregular cell surfaces with pseudopodia, cell shrinkage and breakages in the plasma membrane--typical of apoptosis. TEM indicated that plasma membranes and double nuclear membranes have structural changes, with apoptotic bodies (blebbing) and disrupted mitochondrial membranes. In 17alpha-ethynylestradiol-exposed neurons, disruption of the plasma membrane with cell swelling and vacuolization was present. No apoptotic bodies or budding were noted here. 17beta-Estradiol induced openings in the plasma membrane, while DES-exposed neurons did not show any morphological changes. Therefore we conclude that EDC damage is morphologically visible and the damage is recognized as apoptosis and oncosis. Estrogenic substances may hence modify hormonal actions thereby leaving the developing nervous system more susceptible to damaging events.