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PubMed1991

Dithranol-induced cytotoxicity in primary cultures of rat epidermal keratinocytes. I. The role of reactive oxygen species.

Primary cultured rat epidermal keratinocytes were used as an experimental model to detect oxidant-mediated adverse effects of dithranol (anthralin), a widely used antipsoriasis drug with tumor-promoting and skin-irritating properties. Keratinocytes were isolated and prepared from the skin of neonatal rats by a trypsin flotation method. Highly proliferative monolayer cells cultured in a serum-free medium were exposed to the test compound at concentrations (5-100 microM) used therapeutically for the treatment of skin disorders. Cytotoxicity was evaluated by changes in plasma membrane integrity (lactate dehydrogenase leakage), lysosomal function (neutral red uptake), and mitochondrial metabolic activity (reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, MTT). Exposure of keratinocytes to dithranol produced time- and concentration-related toxic responses. MTT reduction was found to be a more sensitive endpoint of cytotoxicity, showing significant toxic effects at 2 hr, while significant leakage of lactate dehydrogenase did not result until 6 hr. Oxygen consumption in keratinocytes and isolated mitochondria showed a similar pattern after exposure to dithranol. Increased cyanide-insensitive respiration was also noted. Oxidative stress, measured by superoxide anion-dependent reduction of nitroblue tetrazolium, occurred before dithranol produced cytotoxicity in the keratinocyte cultures. Superoxide formation, which increased with time after dithranol exposure, was detected both extracellularly and intracellularly and was inhibited by the addition of superoxide dismutase. Dithranol-induced cell injury was partially prevented by treatment with superoxide dismutase, and greater protection was shown by concurrent treatment with superoxide dismutase plus catalase. These findings suggest that the superoxide anion and hydrogen peroxide may be involved in the cytotoxicity of dithranol and that a culture system of rat keratinocytes may be useful in evaluating the mechanism of toxicity of dermatotoxicants.

Animals↗
PubMed1983

Kinetic analysis of the protonation of a surface group of a macromolecule.

The dynamics of proton transfer between a surface-attached acidic moiety and the bulk of the solution was measured using the laser-induced proton pulse technique. [Gutman, M., Huppert, D. and Pines, E. (1981) J. Am. Chem. Soc. 103, 3709-3713]. The model system for this study consists of pH indicators (either neutral red or bromcresol green) adsorbed on Brij 58 micelles, as defined targets for protonation and a non-adsorbed proton emitter (2-naphthol-3,6-disulfonate) for generation of protons in bulk. The reaction was measured with 50-ns time resolution over a time period of about 200 microseconds. The results were analyzed by a numerical solution of the coupled nonlinear differential equation corresponding with the reaction system. [Gutman, M., et al. (1983) J. Am. Chem. Soc. 105, 2210-2216]. Quantitative analysis reveals two independent reactions which govern the observed dynamics: (a) a diffusion-controlled reaction between the proton and the surface targets; (b) translocation of the protonated target between the hydration layer of the interface and a more hydrophobic one. The contribution of the translocation reaction to the dynamics of surface protonation is more pronounced for compounds like carboxylates or phenolates which increase their hydrophobicity upon protonation. Amines and azoaromatic structures are more hydrophilic in their protonated states, the dynamics of their protonation is less affected by post-protonation distribution within the microenvironments of the interface. The interrelation between the partial rate constants and the macroscopic time constants and equilibrium parameters is analyzed.

Bromcresol Green↗
PubMed1994

Primary culture of rat gastric epithelial cells as an in vitro model to evaluate antiulcer agents.

Primary rat gastric cell cultures were investigated as an in vitro model for evaluating antiulcer agents. Following exposure to concentrations of up to 5 mg/mL of an antiulcer agent sucralfate, an aluminum hydroxide complex of sucrose octasulfate, cultured cells were treated with either pH 3.5 medium or 3.5 mM indomethacin. Cytoprotection was evaluated by colony forming efficiency, neutral red uptake, and 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) hydrolysis. By each measure, and depending on damaging agent, 2 and 5 mg/mL sucralfate provided partial (50% of untreated control) to near-complete (90% of untreated control) cytoprotection, respectively. Aluminum hydroxide also provided partial (55% of untreated control) to near-complete (more than 90% of untreated control) cytoprotection at 2 and 5 mg/mL, respectively, for the pH 3.5 medium-induced damage. Over a concentration range of 0.05 to 5 mg/mL, the potassium salt of sucrose octasulfate, KSOS, stimulated cell growth up to 40-60% over untreated controls but had little or no cytoprotective action in the presence of either 3.5 mM indomethacin or pH 3.5 medium. Overall results suggested that sucralfate may have at least two roles in influencing gastric epithelial cell function, cytoprotection and stimulation of cell growth in vitro. These observations serve as a basis for further study of in vitro models in evaluating the cytoprotective activity of antiulcer agents and their respective mechanisms of action.

Acids↗
PubMed2006

Pathological reactions and recovery of hepatopancreatic digestive cells from the marine snail Littorina littorea following exposure to a polycyclic aromatic hydrocarbon.

The aim of this study was to investigate the cellular pathological responses of hepatopancreatic digestive cells from the periwinkle Littorina littorea exposed to the polycyclic aromatic hydrocarbon (PAH) fluoranthene and to ascertain whether any injurious effects were reversible within the experimental time scale. A secondary objective was to establish the relationship of the various reactions to animal health status, using lysosomal stability as an index of well-being. Exposure of snails to a concentration of 335 microgl(-1) (1.7 microM) fluoranthene (seawater renewed and spiked daily with fluoranthene) for 5 days resulted in a reduction in lysosomal stability (neutral red retention) and endocytosis; and an increase in smooth endoplasmic reticulum (ER) and 7-ethoxycoumarin-o-deethylase (ECOD; measured as cyano-ECOD) activity measured in isolated live digestive cells. Exposed snails treated with clean seawater for a further 8 days resulted in a return to control levels of lysosomal stability, ECOD and ER; endocytosis showed only a partial recovery. Multi-variate and uni-variate analysis showed that there were strong correlations between the various cellular biomarker responses. These findings are interpretable within the current framework of molluscan biomarker responses to PAHs. Principal component analysis was used to derive the first principal component for endocytosis, ER and ECOD reactions and these were plotted against lysosomal stability as a measure of cellular well-being. The resulting significant regression represents the mapping of the individual biomarkers within health status space for a gradient of fluoranthene toxicity. From this analysis, we concluded that endocytosis is an indicator of healthy snails while proliferation of ER and to a lesser extent induced ECOD are indicative of dysfunction and reduced health. Finally, the results indicate that stress induced by chronic exposure to a PAH is reversible.

7-Alkoxycoumarin O-Dealkylase↗
PubMed2004

In vitro assessment of copper-induced toxicity in the human hepatoma line, Hep G2.

Copper, though essential, is highly toxic when present in excess, as in Wilson disease, a genetic disorder of hepatic copper metabolism. We hypothesized that mitochondria are a major target of copper-induced cytotoxicity in Wilson disease. We used the human hepatoma line Hep G2 to examine copper-mediated cytotoxicity and three different methods to assess organelle damage: MTT assay (mitochondria), neutral red (NR; lysosomes) and Trypan blue exclusion assay (TB; plasma membrane). For all assays, cells at approximately 60% confluence in microtitre plates were incubated with CuCl(2) (concentration range: 50-100-150-200 microM) for 24 or 48 h. Results were expressed as percent of untreated control. At 24 h, cytotoxicity as detected by NR assay was significantly higher at all concentrations of copper than for MTT or TB ( p<0.005 at all concentrations). Cytotoxicity as detected by MTT was higher than that detected by TB at all concentrations except at 200 microM (p<0.05 for 50 microM, p<0.005 for 100 microM, p = 0.001 for 150 microM). Results at 48 h were similar (NR versus others: p <0.001 MTT versus TB: NS except at 150 microM where p<0.01). We investigated reactive oxygen species (ROS) production in copper-associated hepatocytoxicity by incubating sub-confluent cells with 2('),7(')-dichlorodihydrofluorescein diacetate dye plus copper (concentration range: 0-200 microM) for 1-1.5 h. Copper, but not zinc, produced significant increases in ROS (p<0.001). In summary, Hep G2 lysosomes appeared more susceptible to Cu-mediated damage than mitochondria; the cell membrane was highly resistant to damage.

Carcinoma, Hepatocellular↗
PubMed2006

In vitro cytotoxicity assessment of the biocidal agents sodium o-phenylphenol, sodium o-benzyl-p-chlorophenol, and sodium p-tertiary amylphenol using established fish cell lines.

The cytotoxicity of three biocidal agents frequently employed as active ingredients in phenolic-based disinfectants, were evaluated in three established fish cell lines (EPC, CHSE and RTG-2). Cell viability was assessed using two fluorescent indicator dyes, Alamar Blue for metabolism and neutral red for lysosomal activity. Total protein content was also quantified as a measure of cell detachment. In order to evaluate the sensitivity of the cell cultures, the results obtained were compared with toxicity data obtained from a previous study with the same three compounds and the in vivo lethality test with rainbow trout. Results from this study established that each of the three cell lines ranked the tested chemicals in the same order of toxicity as the in vivo test; however, the cell cultures were found to be an order of magnitude less sensitive than whole fish studies with the same compounds. The chemical sodium o-benzyl-p-chlorophenol was consistently ranked the most toxic of the tested compounds with each cell line and the endpoints employed. The rank order of toxicity was always sodium o-benzyl-p-chlorophenol > sodium p-tertiary amylphenol > sodium o-phenylphenol. The EPC cells were found to be the most sensitive cell line tested based on Alamar Blue IC(50) data, and the Alamar Blue assay was consistently found to be the most sensitive endpoint of the three cytotoxicity assays employed.

Animals↗
PubMed1980

Mechanism of lethal effect of human serum upon Leishmania donovani.

In order to gain greater understanding of potential host defense mechanisms against Leishmania donovani, we examined the effect of nonimmune, human serum upon promastigotes and amastigotes. Fresh sera were found to be lethal for promastigotes, but had no detectable effect on amastigotes. Serum exposed promastigotes became immotile, did not take up neutral red dye, appeared to be disrupted, and failed to recover after further incubation in fresh media. Heat labile components were required for promastigote killing since heat-inactivated serum (56 degrees C, 30 min) agglutinated but did not kill them. Sera that lacked either the 5th or 6th complement (C) component had no effect when used alone, but when used together, were lethal, indicating that activation of the membrane attack complex (C5b-C9) ws necessary for the lethal effect. The mode of C activation was determined by using serum with complete, selective, deficiency of C2, and normal serum chelated with Mg-EGTA. The C2-deficient serum killed promastigotes only after the addition of purified C2, and Mg-EGTA chelated serum had no detectable lethal effect. Thus, promastigotes appeared to activate C through the classical pathway. Human IgG and IgM, detected with 125I-anti-human antibody, bound to promastigotes. Removal of antibody from serum by absorption with promastigotes eliminated the lethal effect. The effect was restored by addition of heat-inactivated serum to absorbed serum. We conclude that promastigotes bind antibody and are killed by activation of the membrane attack complex of C through the classical pathway.

Animals↗
PubMed1994

In vitro and in vivo viability assessment of unpurified pancreatic islet tissue.

The viability of porcine collagenase-prepared islet preparations (n = 16) was classified by 31P-NMR spectroscopy, staining by neutral red and trypan blue, and in vitro insulin secretion following glucose challenge. Vital islets exhibited a phosphate diester/phosphate monoester (PDE/PME) ratio of 0.5-0.9, a staining score of 18-30 and an insulin secretion responding well to glucose challenge. Damaged islets performed at a PDE/PME of 0.2-0.49 and a staining score of 9-17 and necrotic islets had 0.0-0.49 and a staining score of 9-17 and necrotic islets had 0.0-0.19 and 0-8, respectively. The islets of the latter two groups did not adequately respond to glucose. The in vivo function following autotransplantation of these islets into the spleen was investigated in five recipients of more than 3000/kg vital islets of which 4 expressed daily normoglycemia (< 200 mg%), normalized intravenous glucose tolerance (K = -2.21), and a prolonged survival (mean +/- SD) of 167 +/- 12 days compared to five recipients of > 3000/kg damaged islets (K = -0.814) (P = 0.0017) and a survival of 86 +/- 21 days (P = 0.0096). It is suggested that 31P-NMR spectroscopy is a valuable and practical method to predict islet graft viability prior to transplantation in order to assure good graft function in the recipient.

Animals↗
PubMed2004

Assessment of soil toxicity from an antitank firing range using Lumbricus terrestris and Eisenia andrei in mesocosms and laboratory studies.

Earthworm mesocosms studies were carried out on a explosives-contaminated site at an antitank firing range. Survival of earthworms and the lysosomal neutral red retention time (NRRT), a biomarker of lysosomal membrane stability, were used in these studies to assess the effect of explosives-contaminated soils on the earthworms Lumbricus terrestris and Eisenia andrei under field conditions. Toxicity of the soils samples for E. andrei was also assessed under laboratory conditions using the earthworms reproduction test and the NRRT. Results indicate that the survival was reduced up to 40% in certain explosive-contaminated soil mesocosms following 10 days of exposure under field conditions, whereas survival was reduced up to 100% following 28 days of exposure under laboratory conditions. Reproduction parameters such as number of cocoons and number of juveniles were reduced in many of the selected contaminated soils. Compared to the reference, NRRT was significantly reduced for E. andrei exposed to explosive-contaminated soils under both field and laboratory conditions, whereas for L. terrestris NRRT was similar compared to the reference mesocosm. Analyses showed that HMX was the major polynitro-organic compound in soils. HMX was also the only explosive detected in earthworm tissues. Thus, results from both field mesocosms and laboratory studies, showed lethal and sub-lethal effects associated to soil from the contaminated area of the antitank firing range.

Animals↗
PubMed2003

Phototoxicity and photogenotoxicity of nine pyridone derivatives.

Nine structurally related pyridone derivatives were assayed for photogenotoxicity and phototoxicity in the Ames test, the chromosomal aberration test in V79 cells and the neutral red uptake (NRU) test in 3T3 cells. All nine compounds absorb light to a comparable degree at wavelengths between 380 and 430 nm. Seven of the nine compounds were found to produce high quantities of singlet oxygen (1O(2)) upon irradiation in the presence of oxygen. These seven compounds were highly phototoxic in the NRU test, three were clearly and two were marginally photomutagenic in the Ames test, five were assessed as clearly and two as equivocally photoclastogenic in the chromosomal aberration test. Two compounds showed substantially lower 1O(2) yields. The pyridone ring of these two compounds is attached to a non-aromatic ring, while for the seven other compounds the chromophore system including the pyridone ring consists of two or three aromatic rings. One of the two compounds with low 1O(2) yields was distinctly less phototoxic and did not induce photogenotoxic effects. The other, structurally an indolo derivative and not the common thieno derivative, was, however, similarly phototoxic as the seven compounds with high 1O(2) quantum yield and was also clearly photogenotoxic indicating that different action pathways, not involving singlet oxygen, have to be considered at least for this compound.

3T3 Cells↗
PubMed2003

Hsp27, Hsp70, and metallothionein in MDCK and LLC-PK1 renal epithelial cells: effects of prolonged exposure to cadmium.

Cadmium is a widely distributed industrial and environmental toxin. The principal target organ of chronic sublethal cadmium exposure is the kidney. In renal epithelial cells, acute high-dose cadmium exposure induces differential expression of proteins, including heat shock proteins. However, few studies have examined heat shock protein expression in cells after prolonged exposure to cadmium at sublethal concentrations. Here, we assayed total cell protein, neutral red uptake, cell death, and levels of metallothionein and heat shock proteins Hsp27 and inducible Hsp70 in cultures of MDCK and LLC-PK1 renal epithelial cells treated with cadmium for 3 days. Treatment with cadmium at concentrations equal to or greater than 10 microM (LLC-PK1) or 25 microM (MDCK) reduced measures of cell vitality and induced cell death. However, a concentration-dependent increase in Hsp27 was detected in both cell types treated with as little as 5 microM cadmium. Accumulation of Hsp70 was correlated only with cadmium treatment at concentrations also causing cell death. Metallothionein was maximally detected in cells treated with cadmium at concentrations that did not reduce cell vitality, and further increases were not detected at greater concentrations. These results reveal that heat shock proteins accumulate in renal epithelial cells during prolonged cadmium exposure, that cadmium induces differential expression of heat shock protein in epithelial cells, and that protein expression patterns in epithelial cells are specific to the cadmium concentration and degree of cellular injury. A potential role for Hsp27 in the cellular response to sublethal cadmium-induced injury is also implicated by our results.

Actins↗
PubMed1988

Acute cytotoxicities of polynuclear aromatic hydrocarbons determined in vitro with the human liver tumor cell line, HepG2.

The neutral red in vitro cytotoxicity assay was adapted for use with the human hepatocellular tumor cell line HepG2 to detect the cytotoxic potencies of polynuclear aromatic hydrocarbons (PAHs). Using benzo[a]pyrene (B[a]P) as the representative PAH, it was determined that a 3-day exposure was the most suitable for detecting cytotoxic potency and that preexposure to 5 micrograms/ml Arochlor enhanced the sensitivity of the HepG2 cells to the toxicant. Such enhanced sensitivity probably reflected increased metabolic conversion of the B[a]P to active metabolites after culturing the cells in the presence of Arochlor. This was shown by a 3-fold increase in the activity of 7-ethoxycoumarin deethylase, an indicator of mixed-function oxygenase activity. Furthermore, a reduction in sensitivity to B[a]P occurred when the cells were cultured in the presence of alpha-napthoflavone, an inhibitor of aryl hydrocarbon hydroxylase activity. When Arochlor-induced cells were transferred to medium lacking Arochlor, the level of 7-ethoxycoumarin deethylase quickly declined to basal levels. Arochlor-induced cells were also able to detect the cytotoxic potencies of benzo[k]fluoranthene, benzo[b]-fluoranthene, chrysene, benzo[a]anthracene pyrene, phenanthrene, and fluoranthene, whereas fluorene, anthracene, acenaphthene, and acenaphthylene were not cytotoxic.

7-Alkoxycoumarin O-Dealkylase↗
PubMed2005

Ecotoxicological evaluation of the antimalarial drug chloroquine.

There is limited information available about the potential environmental effects of chloroquine (CQ), a widely used antimalarial agent and a promising inexpensive drug in the management of HIV disease. The acute effects of CQ were studied using four ecotoxicological model systems. The most sensitive bioindicator was the immobilization of the cladoceran Daphnia magna, with an EC50 of 12 microM CQ at 72 h and a non-observed adverse effect level of 2.5 microM CQ, followed very closely by the decrease of the uptake of neutral red and the reduction of the lysosomal function in the fish cell line PLHC-1 derived from the top minnow Poeciliopsis lucida, probably due to the selective accumulation of the drug into the lysosomes. There was significant cellular stress as indicated by the increases on metallothionein and glucose-6P dehydrogenase levels after 24 h of exposure and succinate dehydrogenase activity mainly after 48 h. No changes were observed for ethoxyresorufin-O-deethylase (EROD) activity. The least sensitive model was the inhibition of bioluminescence in the bacterium Vibrio fischeri. An increase of more than five-fold in the toxicity from 24 to 72 h of exposure was observed for the inhibition of the growth in the alga Chlorella vulgaris and the content of total protein and MTS tetrazolium salt metabolization in PLHC-1 cells. At the morphological level, the most evident alterations in PLHC-1 cultures were hydropic degeneration from 25 microM CQ after 24h of exposure and the presence of many cells with pyknotic nuclei, condensed cytoplasm and apoptosis with concentrations higher than 50 microM CQ after 48 h of exposure. In conclusion, CQ should be classified as harmful to aquatic organisms.

Analysis of Variance↗
PubMed2000

In vitro testing of ten bone cements after different time intervals from polymerization.

Biological response of cells to implanted bone cement is a fundamental but often neglected issue in successful cemented implants. In this study, ten acrylic bone cements for orthopedics were assayed using two different in vitro testing methods on L929 cells. The cements were mixed as prescribed, cured for either 1 h or 7 days and then extracted in minimum essential medium (MEM) according to the ISO standard for the preparation of samples. For the evaluation of cytotoxicity, the neutral red uptake assay (NRU) and the incorporation of propidium iodide (PI) were used to detect the viability/death of cells. The two methods were shown to be well correlated (p < 0.0001) in the case of both the 1-h and the 7-day extracts. Two cements, i.e. CERIM LT and CMW2, were found to be toxic after 1-h curing through both the spectrophotometric NRU assay and the cytofluorometric assay with PI. After 7-day curing, these two cements, as well as the Zimmer-low viscosity cement, were toxic according to the NRU assay. The toxic effect of all the cements disappeared after dilution of extracts 1:2 with MEM, except in the case of CERIM LT. In the search for the component inducing the toxic effect, the possible contribution of the residual monomer was discarded on the basis of literature data and the influence of various other factors was analyzed, including the contrast medium (barium sulphate or zirconium dioxide) and the concentrations of N,N-dimethyl-paratoluidine and of benzoyl peroxide (< 1% or > or = 1%). Unlike zirconium dioxide, barium sulphate was found to damage the cells at the 1-h endpoint. Benzoyl peroxide at concentration > or = 1% was found to affect cells at the same endpoint, whereas dimethylparatoluidine had no effect regardless of the proportion.

Animals↗
PubMed1995

Osteoblast adherence and resorption activity of isolated osteoclasts on calcium sulphate hemihydrate.

The adherence of osteoblast-like cells and the resorption activity of isolated osteoclasts on calcium sulphate hemihydrate (CSH) was investigated. After a 24 h incubation period, alkaline phosphatase staining showed that rat osteoblast-like cells ROS 17/2.8 attached on CSH. Neutral red (NR) and tartrate-resistant acid phosphatase (TRAP) staining revealed that osteoclasts attached on CSH. Furthermore, osteoclasts formed lacunae as revealed by scanning electron microscopy. Although the lacunae formed by osteoclasts on CSH were diverse in shape and form, the most common type had approximately a circular outline, with a well-defined margin similar to those formed on dentine. Less commonly, excavations appeared as a discontinuous area of resorbed CSH with the presence of a circular zone around the non-resorbed area. Finally, by using 10(-9) M calcitonin, evidence was obtained that NR-positive cells were osteoclasts (58.3% and 57.66% decrease of NR-positive mouse and rat cells detected on CSH after 24 h incubation). However, no inhibition was obtained with 10(-11) M calcitonin. The overall number of NR-positive osteoclasts adherent on 256 mm2 CSH was 43 +/- 14 and 42 +/- 3 for mice and rat, respectively. The overall number of TRAP-positive mouse osteoclasts was 67 +/- 12. Acetazolamide (10(-5) M), a carbonic anhydrase inhibitor, inhibited the number of adherent NR osteoclasts on CSH by 50.42% and 41.6% for mouse and rat, respectively. These results indicate that osteoblasts attach on CSH and osteoclasts resorb CSH in vitro.

Acetazolamide↗
PubMed2005

Assessing the potential of fish cell lines as tools for the cytotoxicity testing of estuarine sediment aqueous elutriates.

In the present study, we assess the potential of fish cell lines (CHSE, EPC and RTG-2) to be used as screening tools for the ecotoxicological assessment of estuarine sediments. The processing of sediment to a form suitable for in vitro exposure is an inherent problem when using cell cultures. The approach employed in this study was to prepare aqueous elutriate extracts from whole sediments, which were subsequently used to reconstitute powdered media. This procedure allowed the exposure of cell cultures to concentrations of up to and including 100% of the original aqueous sample. Cytotoxicity was assessed using multiple endpoint measurements. Cell viability was quantified using the neutral red and alamar blue colorimetric assays, which specifically assess lysosomal and mitochondrial function, respectively. In addition, the total protein content of the cells was measured using the coomassie blue assay. Initial tests were conducted to ensure that any resultant cytotoxicity was due to sample contaminants and not osmotic stress. In addition, elutriate samples were spiked with a model toxicant to verify the ability of the cell lines to detect and respond to bioavailable contaminants. Chemical analyses were conducted on sediments from all sampling sites to assist in interpreting any observed cytotoxicity. A differential response was observed for the cytotoxicity assays following exposure treatments, which emphasises the importance of employing multiple endpoints for the determination of toxicity. Of the three cell lines utilised in this study, RTG-2 cells were the most suitable for the testing of estuarine aqueous elutriate samples on the basis of tolerance to osmolality effects. Slight toxicity was observed following exposure to the aqueous elutriates tested in this study using RTG-2 cells and the alamar blue assay. In order to fully evaluate the overall sensitivity of this cell line, further research is warranted using an extensive range of test sites incorporating more polluted sediments.

Animal Testing Alternatives↗
PubMed1978

Cytoplasmic vacuoles of Rous virus transformed cells are organelles involved in cation uptake.

Cytoplasmic vacuoles induced during transformation of cells by Bryan strain Rous sarcoma virus (RSV-BH) have been studied using the cationic dye, neutral red(NR). Both the rate of uptake and the accumulation of NR are greater in RSV-BH transformed cells than non-transformed cells however, uptake was greater in vacuolated than in non-vacuolated cells, whether or not they were transformed. The NR was incorporated into pre-existing vacuoles in the absence of cytoplasmic staining, suggesting the existence of direct channels from the cell surface to the vacuoles. Other low mol. wt. cationic dyes could also be incorporated into vacuoles, although those with branched structures or cationic weights greater than 330 were excluded. No anionic dyes were incorporated. Infection of cells with a virus mutant, RSV-BH-Ta, induces temperature-dependent vacuolization. After a shift to the vacuole-permissive temperature, vacuoles developed at different rates and with morphological variations with different cations. Vacuoles which had formed in the presence of several cations, (K+, Rb+, tris+, choline+) failed to disappear when cells were incubated at a temperature sufficient to revert vacuoles formed in Na+-containing medium. No short-term effects of Cl-replacements (Br-, I-, or SO2-4) on vacuolization or reversal were observed. The results suggest that these vacuoles are organelles involved in cation uptake. A possible function for these organelles in RSV-BH induced malignancy is discussed.

Animals↗
PubMed1990

Location of osteoclast precursors in fetal rat calvaria cultured on collagen gels.

Although osteoclasts are derived from hematopoietic cells, the exact identity of their precursors and the mechanism for their recruitment onto bone surfaces remain unclear. We wished to study their differentiation in the fetal rat calvaria and to locate its source of osteoclast precursor cells. Osteoclasts were detected by neutral red staining or cytochemical reaction for acid phosphatase of intact bone (cell number and area measured by computerized image analysis) or in cryostat sections of bone (enzyme activity measured by quantitative cytochemistry). Histology of semithin sections of fixed bones was also examined. The 19 day calvariae contained few mature osteoclasts. After 48 h culture on gels of type 1 collagen (1.5 mg/ml) supplemented with 5 mM calcium beta-glycerophosphate, 10 mM proline, and 2 micrograms/ml ascorbic acid, numerous large osteoclasts were seen on their endocranial surfaces. In contrast, cell morphology and enzyme activity deteriorated in bones cultured in liquid medium. The cells that formed in vitro rapidly responded to calcitonin by contraction. Stripping of endocranial membranes from the calvariae prevented osteoclast formation in culture, but these cells were seen when "stripped" bones had been cocultured with their membranes for 48 h or with intact 16 day calvariae (well before the onset of osteogenesis). Few osteoclasts were found when an 0.22 micron filter was inserted between the stripped calvaria and the endocranial membranes. We conclude that the endocranial membranes, which contain the meningeal blood vessels, are a major source of osteoclast precursors and that these cells are present in calvarial tissue even before the onset of osteogenesis.

Acid Phosphatase↗