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At least 739 records · Page 41Linked to original sources
PubMed1996

Flavopiridol: a cytotoxic flavone that induces cell death in noncycling A549 human lung carcinoma cells.

Flavopiridol (NSC 649890, L86-8275), a potent inhibitor of cyclin-dependent kinase 1/p34cdc2 phosphorylation and kinase activity, is currently undergoing Phase I clinical testing as a potential antineoplastic agent. Previous studies have suggested that flavopiridol is cytostatic but not cytotoxic when applied to exponentially growing cells. In the present study, various human tumor cell lines were assayed for trypan blue exclusion and ability to form colonies after exposure to flavopiridol under a variety of growth conditions. When log phase A549 non-small cell lung cancer cells were examined 72 h after the start of a 24-h flavopiridol exposure, as many as 90% of the cells accumulated trypan blue. A 24-h exposure to 250-300 nM resulted in trypan blue uptake in 50% of A549 cells at 72 h and a 50% reduction in colony formation. Similar results were observed in HCT8 ileocecal adenocarcinoma, T98G glioblastoma, MCF-7 breast adenocarcinoma, and HL-60 leukemia cells. With A549 cells, identical results were obtained in actively growing logarithmic phase cells and growth-arrested confluent cells. Treatment with the DNA synthesis inhibitor aphidicolin only minimally affected the cytotoxicity of flavopiridol. In contrast, the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole or the protein synthesis inhibitor cycloheximide reduced the cytotoxicity of flavopiridol. These results suggest that: (a) flavopiridol is not only cytostatic, but also cytotoxic to a variety of human tumor cell lines; (b) flavopiridol is equally active against cycling and noncycling A549 cells; and (c) RNA and protein synthesis appear to play a role in flavopiridol-induced cytotoxicity.

Antineoplastic Agents↗
PubMed1994

Identification of subcellular targets and sensitive tests of ethanol-induced damage in isolated rat gastric mucosal cells.

BACKGROUND/AIMS: The mechanisms of chemically induced injury are poorly understood. The purpose of this study was to develop a rapid method for isolation of gastric mucosal cells (GMCs) and sensitive tests for investigating cell damage and protection. METHODS: GMCs were prepared from rat glandular stomach with pronase and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and kept in albumin-containing, albumin-free, or Hank's solutions. Initial cell viability and responsiveness were tested with trypan blue exclusion, and stimulation or inhibition of pepsinogen secretion were tested with carbachol, 16,16-dimethyl prostaglandin E2, or carbachol plus atropine. Plasma membrane damage was assessed by trypan blue exclusion, lactate dehydrogenase or cell protein leakage, mitochondrial integrity by succinic dehydrogenase activity, and nuclear lesions by ethidium bromide DNA fluorescence. RESULTS: Incubation of GMC with ethanol resulted in 50% lethal dose for ethanol values of 12.3%, 7.4%, and 10.9% by trypan blue exclusion; 10.1%, 9.4%, and 9.1% by lactate dehydrogenase leakage; 12.8%, 10.0%, and 10.4% by succinic dehydrogenase; and 14.1%, 13.0%, and 11.5% by ethidium bromide DNA by binding using albumin-containing, albumin-free, and Hank's solutions, respectively. CONCLUSIONS: Rat GMC with high and long viability can be isolated by low concentrations of pronase and EGTA. The adapted biochemical and microscopic methods are suitable for parallel examination of chemical injury of plasma membrane, mitochondria, and nuclei. Albumin increased ethanol 50% effective concentration to GMC. A combination of biochemical and morphological methods with ethanol-induced injury to GMC is proposed for direct cytoprotection investigations.

Animals↗
PubMed1992

Cell-growth quantitation methods for the evaluation of antiestrogens in human breast cancer cells in culture.

The antiproliferative activity of the antiestrogen, tamoxifen, on the growth of MCF-7 human breast cancer cells was evaluated using the hemocytometric trypan blue exclusion method, [3H]-thymidine incorporation, and a total protein determination. Tamoxifen was evaluated over a concentration range from 10(-9) to 10(-6) M. The hemocytometric trypan blue exclusion method and [3H]-thymidine incorporation were sensitive enough to demonstrate the inhibitory influence of tamoxifen on the proliferation of MCF-7 cells at a concentration as low as 10(-9) M. A very good correlation of these two methods was observed in the submicromolar concentration range of tamoxifen. The total protein determination method was only sensitive enough to detect the antiproliferative influence of tamoxifen at concentrations above 10(-6) M. In conclusion, the [3H]-thymidine incorporation method was found to be effective and much less time consuming than the hemocytometric trypan blue exclusion method for evaluating the antiproliferative effects of antiestrogens in cultured MCF-7 cells. However, when evaluating antiestrogens, which are cell-cycle specific, the results of the [3H]-thymidine incorporation method should be interpreted with caution.

Algorithms↗
PubMed1998

Mechanisms of cytotoxic effects of heavy water (deuterium oxide: D2O) on cancer cells.

Heavy water (deuterium oxide: D2O) contains a neutron and a proton in its hydrogen atoms and shows a variety of biologic activities different from normal light water. In the present study the cytotoxic and cytostatic activity of D2O was assessed using a BALB/c-3T3 fibroblast cell line and four human digestive organ cancer cell lines, i.e. HepG2 hepatic, Panc-1 pancreatic, KATO-3 gastric and Colo205 colonic cancer cell lines. Against four cancer cell lines, D2O showed significant cytotoxic and cytostatic effects in a MTT assay and a Trypan blue dye exclusion assay, at concentrations higher than 30% D2O. These effects were time and dose dependent, and the IC50 after 72 h of culture ranged from 20 to 30% D2O in the Trypan blue dye exclusion assay and from 30 to 50% D2O in the MTT assay. By contrast, IC50 for the 3T3 fibroblast cell line after 72 h of culture was about 15% in the Trypan blue dye exclusion assay and 50% inhibition was not achieved in the MTT assay. Furthermore, D2O was found to significantly inhibit the invasion of tumor cells in a Matrigel invasion chamber assay at concentrations higher than 10% D2O. Incubation with D2O resulted in enlargement of cells, nuclear pyknosis and vacuolization, and immunostaining studies demonstrated that D2O treatment resulted in an increase in nuclear nick-end-labeling, which indicates DNA fragmentation, in KATO-3 and HepG2 cell lines. Furthermore, the nucleic acids and protein synthesis inhibition assay suggested that the inhibition of DNA synthesis may be one of the mechanisms responsible for the antitumor effects of D2O. Furthermore, oral administration of D2O resulted in a significant inhibition of the growth of Panc-1 tumor xenografted s.c. in nude mice, but survival was not prolonged. In conclusion, D2O has cytotoxic and cytostatic activities against human digestive organ cancer cell lines, and D2O may be a potential anticancer agent.

3T3 Cells↗
PubMed1998

Assessment of hair cell death using the dye extrusion method.

We examined the usefulness of the dye extrusion method for assessing cochlear hair cell death in guinea pigs. Unilateral temporal bones were incubated in sterile Dulbecco modified Eagle's medium for 1-6 h and then perfused with PBS/0.3% trypan blue to determine hair cell viability. Cells with nuclei stained with trypan blue were assumed to have died. The contralateral temporal bones of each animal were incubated with the same medium for 1-6 h and examined by transmission electron microscopy (TEM). This method revealed that hair cell viability under these conditions was maintained for at most 3 h. After incubation for more than 3 h, hair cell injury began and increased. After 6 h incubation, TEM revealed apoptotic death of outer hair cells. The results with this method agreed with the TEM findings. The dye extrusion method is simple, easy and useful for screening hair cell death in the cochlea under pathological conditions.

Animals↗
PubMed1984

Assessment of cell damage caused by spontaneous lipid peroxidation in rabbit spermatozoa.

Damage to the plasma membrane of rabbit epididymal spermatozoa during spontaneous lipid peroxidation was examined by means of trypan blue uptake and expression of activity of the intracellular enzymes, lactate dehydrogenase and pyruvate kinase. Both the dye uptake and the expression of enzyme activity probe cell damage from lipid peroxidation as loss of integrity of the plasma membrane. A linear correlation was obtained between trypan blue staining of the cells and malondialdehyde production, a quantifiable measure of the extent of lipid peroxidation. At the point of trypan blue staining of all cells, 0.5 nmol malondialdehyde/10(8) cells was produced. This is the same amount produced at the point of complete loss of motility and superoxide dismutase activity. We have defined this as the "lipoperoxidative lethal end point." Expression of lactate dehydrogenase and pyruvate kinase activities increased with time of aerobic incubation. In the high Na+ medium, NTP, in which lipid peroxidation is slow, there is a linear correlation between increase in expressed enzyme activities and malondialdehyde production. But in the high K+ medium, KTP, in which lipid peroxidation is rapid, there is an initial rapid rise in expressed enzyme activity over 3 h, followed by a slower increase. Activities of rabbit sperm lactate dehydrogenase, pyruvate kinase, and flagellar ATPase were unaffected by aerobic incubations for up to 48 h, double the incubation period used for the assay of enzymatic activities for the first two. The activity of glyceraldehyde-3-phosphate dehydrogenase decreased during aerobic incubation, the time course matching the loss of motility. The subcellular distribution of lactate dehydrogenase in rabbit spermatozoa was determined: 4% in the mitochondrial matrix, 10% in the plasma membrane and 85% in the cytosolic compartment.

Adenosine Triphosphatases↗
PubMedDate not supplied

Lysis and necrosis: analysis of two cytotoxic phenomena mediated by lymphocytes.

Stimulation of rat lymphocytes by allogeneic and xenogeneic (mouse) embryo fibroblast monolayers or by pokeweed mitogen (PWM) generate two different cytotoxic manifestations: a) cell-mediated target cell lysis obtained by contact between effector cells and target cells; b) cytotoxicity caused by lymphotoxin (here termed "necrosis"). Activation of hypersensitive lymphocytes by soluble antigen, e.g., keyhole limpet hemocyanin (KLH), produces only necrosis but no lytic effect. This difference between mitogen and antigen activation is attributed to the affinity of the mitogens for target cell membranes. There is a marked difference in the time of onset of the two phenomena. Ten minutes after the effector cells have been introduced to the target monolayers, lysis of fibroblasts becomes visible, whereas necrosis usually develops only 48 to 72 h later. A clear difference in the morphology of the two types of target cell killing was seen when the cultures were stained in situ with trypan blue. Lysed target cells produced blebs and underwent fragmentation, scattering cytoplasmic droplets. During a 10-min to 24-h period, these cells excluded the dye. Only much later (after greater than 30 h), when the cell remnants had undergone a further slow "decay" process, did they stain with trypan blue. In cultures rendered necrotic by lymphotoxin, the killed fibroblasts preserved their morphological integrity but the nuclei became markedly stained with trypan blue. Lymphotoxin was found to be effective only in highly concentrated macrophage and fibroblast cultures. In sparsely populated macrophage cultures, the mediator(s) did not effect cell death but nonetheless exerted marked morphological changes. Vacuoles containing lipids developed and eventually occupied the greater volume of the cytoplasm. Giant lipid droplets were seen leaking out of the macrophage cytoplasm. It is suggested that lymphotoxin does not kill directly, but rather that metabolic products released by the vigorously stimulated target cells cause the necrotic effect in the cultures.

Animals↗
PubMed2004

Ultrastructural study of pleural lymphatic drainage unit and effect of nitric oxide on the drainage capacity of pleural lymphatic stomata in the rat.

The objective of this study was twofold: first to investigate the ultrastructure of the lymphatic drainage unit on the costal pleura of rats by electron microscopy, and secondly to examine the effect of nitric oxide on the pleural lymphatic stomata and fluid absorption from the pleural cavity. The lymphatic drainage unit of the rat costal pleura is composed of three special components: the lymphatic stomata between the mesothelial cells, the initial part of the lymphatic vessels and the underlying connective tissue containing many foramina. The unit is the main passage to drainage fluid, particles and cells in the pleural space. To investigate the regulator of the lymph drainage, nitric oxide synthase inhibitor and nitric oxide donor were injected into the peritoneal cavity of the rats, respectively. Trypan blue was used as tracer. The ultrastructural changes of pleural lymphatic stomata were observed under scanning electron microscope and analyzed by a computer image processing system. It turned out that the area and density of the pleural lymphatic stomata were positively correlated with the nitric oxide quantity (p < 0.05). After the tracer was injected into the pleural cavity, the nitric oxide donor group exhibited a higher trypan blue concentration than the control group (p < 0.05). The ability of the pleura to absorb trypan blue was enhanced because of the larger opening of the lymphatic stomata (p < 0.05). It is suggested that nitric oxide can increase lymphatic absorption of the pleura by opening pleural lymphatic stomata.

Animals↗
PubMed2000

Suppression by eicosapentaenoic acid of oxidized low-density lipoprotein and lysophosphatidylcholine-induced migration in cultured rat vascular smooth muscle cells.

The migration of medial smooth muscle cells into the intima is proposed to be an initial process of intimal thickening in atherosclerotic lesions. The present study was designed to determine whether pretreatment with the antiatherogenic agent eicosapentaenoic acid (EPA) inhibits the migration induced by oxidized low-density lipoprotein (LDL) and its major phospholipid component, lysophosphatidylcholine (lyso-PC), in cultured rat vascular smooth muscle cells (VSMCs) using Boyden's chamber method. The effects of EPA pretreatment on angiotensin II (Ang II)- and platelet-derived growth factor BB (PDGF BB)-induced migration were also examined in these cells. Oxidized LDL and lyso-PC induced migration in a concentration-dependent manner. EPA pretreatment clearly suppressed oxidized LDL (200 microg/mL)- and lyso-PC (10(-5) mol/L)-induced migration between 40 and 160 micromol/L. EPA pretreatment also suppressed Ang 11 (10(-7) mol/L)- and PDGF BB (5 ng/mL)-induced migration at a concentration of 80 and 160 micromol/L. However, in a trypan blue exclusion test, dead cells stained with trypan blue were not found 24 hours after treatment with EPA. These results suggest that EPA suppresses VSMC migration induced by oxidized LDL and lyso-PC, as well as Ang II and PDGF BB. These preliminary data concerning the effects of EPA may partly explain the antiatherosclerotic effects of this agent.

Animals↗
PubMed1994

[Vital preservation of cartilage transplants with tissue culture methods].

Cartilage grafting is one of the most common procedures in plastic surgery. Since storage of both autologous and allogenic cartilage is necessary, different preservation methods have been used with more or less success. The use of chemical preservation procedures like formaldehyde, Merthiolate or Cialit lead to a loss of the vitality of the graft. This work presents a study of the cell vitality and the matrix of cartilage grafts stored in different solutions (formaldehyde, saline, RPMI 1640, Ham F-12 and DMEM 4500) during 150 days. The cell viability was assessed using tissue sections (neutral red supravital staining and trypan blue dye exclusion test) and isolated cells (trypan blue dye exclusion test and cell adhesion in monolayer culture). The state of the cartilage matrix was analysed by means of the azan, alcian and toluidine blue staining). Cartilage immersed in formaldehyde solution lost 100% of the vitality after a storage period of 10 days, the one immersed in saline solution after 30 days. Cartilage stored in tissue culture media retained its vitality (> 85%) during the whole storage time. Histological staining methods showed a decrease of the staining intensity after 10 days storage in formaldehyde and after 30 days storage in saline solution. No differences in vitality and the matrix staining were found among all three culture media. Our results suggest that viable cartilage tissue can be successfully stored for a long time using tissue culture methods.

Cartilage↗
PubMed2002

Inhibition of migration and proliferation of rat vascular smooth muscle cells by a new HMG-CoA reductase inhibitor, pitavastatin.

The migration and proliferation of vascular smooth muscle cells (SMCs) are known to play roles in the pathogenesis of atherosclerosis. Therapy with a reductase inhibitor of 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) ("statin") produces significant alterations in various SMC functions. The objectives of the present study were to determine whether pitavastatin, a new chemically synthesized and powerful statin, can affect angiotensin II (Ang II)- and platelet-derived growth factor (PDGF)-induced migration and proliferation of cultured rat vascular SMCs. The effect of pitavastatin on cell viability was also examined in these cells. Migration was evaluated by the Boyden's chamber method using microchemotaxis chambers. As expected, Ang II and PDGF BB potently stimulated cell migration in a concentration-dependent manner. Pitavastatin significantly inhibited Ang II (10(-6) mol/l)-induced migration at the concentrations of 10(-8) and 10(-7) mol/l. Pitavastatin also inhibited PDGF BB (1 ng/ml)-induced migration at concentrations between 10(-9) and 10(-8) mol/l in a relatively concentration-dependent manner. This statin modestly but significantly inhibited Ang II (10(-6) mol/l)- and PDGF BB (1 ng/ml)-induced DNA synthesis at concentrations between 10(-9) and 10(-7) mol/l. In addition, pitavastatin clearly inhibited Ang II (10(-6) mol/l)- and PDGF BB (1 ng/ml)-induced increases of cell number at concentrations between 10(-9) and 10(-7) mol/l. Pitavastatin did not affect lactate dehydrogenase release from these cells at the concentrations used in this experiment. In a trypan blue exclusion test, dead cells stained with trypan blue were not found 24 h after treatment with 10(-9), 10(-8) or 10(-7) mol/l of pitavastatin. These findings suggest that pitavastatin suppresses the migration and proliferation stimulated by Ang II and PDGF BB without affecting cell viability. Pitavastatin may exert an anti-atherogenic effect, in part, through these mechanisms.

Animals↗
PubMed1995

Flow-cytometric studies of the phagocytic capacities of equine neutrophils.

Methodological aspects of flow-cytometric evaluation of the phagocytic properties of equine neutrophils were elucidated. The kinetics of attachment and ingestion were studied, and the phagocytic process was more rapidly completed when serum-opsonized yeast cells were used than with use of IgG-opsonized yeast cells. Trypan blue was successfully used to quench fluorescence of non-ingested yeast cells. There were only minor differences in the kinetics of phagocytosis between quenched and unquenched samples, indicating that attachment is rapidly followed by ingestion. Trypan blue quenching caused loss of cells with light scattering properties of granulocytes, although this did not affect the determined frequencies of truly phagocytic neutrophils. Aggregation of yeast cells proved to be a disturbance but not an obstacle to the determination of frequencies of actively phagocytic cells. Flow cytometry is well suited for studies of phagocytosis of yeast cells by equine neutrophils, and the trypan blue quenching provides a means of eliminating false-positive events due to aggregation of yeast cells. The main advantage of the flow-cytometric method is the possibility of rapid processing of a large number of samples, making the method useful for studies of herds.

Animals↗
PubMed1997

A rapid in vitro assay for evaluation of metabolism-dependent cytotoxicity of antiepileptic drugs on isolated human lymphocytes.

In vitro assessment of human lymphocyte viability by trypan blue dye exclusion in the presence of an external metabolizing system (microsomes plus NADPH) has been shown to be a useful method in assessing predisposition to idiopathic toxicity in response to various anticonvulsant drugs. The trypan blue method, however, is labor intensive, is time consuming, is prone to human error, is not suitable for high-volume toxicity screening, and excludes autolysed cells. The objective of this study was to develop a rapid, high-capacity, objective, and easy in vitro cytotoxicity method for the detection of metabolism-dependent cytotoxicity of a test chemical. The in vitro system uses an external metabolizing system (rabbit microsomes) in conjunction with isolated human lymphocytes as the target cells. Cellular toxicity was determined by assessing plasma membrane integrity using a membrane-impermeant fluorescent nucleic acid dye (YO-PRO-1) and a multiwell plate scanner for fluorescence. Using this system, cells incubated with either acetaminophen (1500 micrograms/ml), carbamazepine (62.5 microM), phenytoin (62.5 microM), or phenobarbital (62.5 microM) showed net increases in percentage cell death of 31 +/- 5, 11 +/- 4, 0 +/- 3, and 2 +/- 3, respectively. A metabolism-dependent concentration-response was observed for valproic acid-induced cytotoxicity, which approached a plateau at a concentration of 4000 micrograms/ml with a net percentage cell death of 31 +/- 4. This technique resolves various technical difficulties inherent in viability determinations by the trypan blue exclusion method. The YO-PRO-1 method also may be useful in a clinical setting for the assessment of patients with a genetically determined susceptibility to certain drugs and for identifying the responsible drug in patients with idiopathic toxicity undergoing multiple-drug therapy.

Acetaminophen↗