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PubMed1981

Neuroglia in amphibian (Rana tigrina) central nervous system.

Neuroglia in the vertebrates are primarily classified as (i) Ependymal and (2) Non-ependymal gliocytes. In the present study neuroglia in the brain and spinal cord of Frog (Rana tigrina) has been studied under the light microscope using rapid Golgi, Holzer, and Luxol fast blue-neutral red staining methods. Ependymal glial cells with long processes having vascular and/or superficial terminal contacts, non-ependymal primitive glial cells (unipolar and bipolar) and phylogenetically more mature non-ependymal glial cells conforming to the classical description of astrocytes, oligodendrocytes and microgliocytes have been observed. It is interesting to note that oligodendrocytes of classical morphology have been observed in close relation to non-myelinated nerve fibres suggesting that nerve fibre-oligodendrocytes relationship may also be other than myelinogenesis.

Animals↗
PubMed1981

Possible requirement of internalization in the mechanism of in vitro cytotoxicity in tumor necrosis serum.

The mechanism of in vitro cytotoxicity by tumor necrosis serum (TNS) and a purified fraction was examined using sensitive L-M cells. Cell death was assessed by uptake of the dye trypan blue and/or by release of radiolabeled chromium. Cell killing was time and dose dependent. Cell survival was assessed by counting the number of survivors via their ability to internalize the dye neutral red and/or by adherent cell protein. The cytotoxin was not cytostatic. Survival was found to be inversely proportional to cell death. The change in survival was used to estimate the number of cells killed. The number of cells killed was logarithmically related to the amount of toxin. The ability to kill a fixed number of cells was inversely related to the number of cells in the assay well. It was estimated that, at ideal cell seed numbers, 1 microgram of TNS protein per 250 microliter killed about 50,000 cells in a 20-hr period. Sensitivity was equated with the amount of TNS required to kill 35,000 cells in 20 hr. Inhibitors of RNA and protein synthesis and also elevated temperatures enhanced sensitivity. The combined treatment of 1 microM actinomycin D and 40 degrees enhanced sensitivity by 15-fold. Resistant normal and tumorigenic cell lines (including human) were rendered sensitive by concomitantly treating them with TNS and cycloheximide or actinomycin D. Cytoskeletal-disrupting agents (colchicine, Colcemid, and cytochalasin B), inhibitors of lysosome activity (chloroquine, methylamine, and leupeptin), and 32 degrees all depressed sensitivity. Sensitivity was nearly equivalent in calcium-free medium. Results substituting partially purified cytotoxin were similar. The results suggest that a toxic factor(s) may need to be internalized and that lysosomal activity may be necessary for cell killing. Comparisons with lymphotoxin are discussed.

Animals↗
PubMed1995

Imidocarb residues in edible bovine tissues and in vitro assessment of imidocarb metabolism and cytotoxicity.

Imidocarb residues in liver and muscle were measured by HPLC after a single therapeutic dose to cattle (3 mg imidocarb dipropionate kg-1). Imidocarb and 7-ethoxycoumarin metabolism were compared in three different in vitro systems prepared from bovine liver: cultures of hepatocyte monolayers, precision-cut liver slices, and microsomes. The potential hepatotoxicity of imidocarb residues was tested on hepatocyte monolayers and assessed using the neutral red and lactate dehydrogenase leakage assays. The concentration of imidocarb (mean +/- SD) decreased between days 14 and 224 after treatment from 5.40 +/- 0.61 to 0.12 +/- 0.01 and from 1.05 +/- 0.31 to 0.06 +/- 0.02 microgram g-1 in liver and muscle, respectively. The depletion kinetics of imidocarb fitted a two-compartment model with alpha- and beta-phase half-lives of 31.7 and 48.5 days in liver and 34.9 and 120.7 days in muscle, respectively. Imidocarb metabolites were not detected in any in vitro system. 7-Ethoxycoumarin metabolism was found in all in vitro systems; the predominant metabolite produced by hepatocyte and liver slice cultures was umbelliferone glucuronide. Cytotoxicity of imidocarb (100 microM) to hepatocyte monolayers was maximal after 72 hr treatment and dose-dependent above 10 microM imidocarb. It is most likely that the hepatotoxicity of imidocarb is caused by the parent compound, because no evidence for imidocarb metabolism was found.

Animals↗
PubMed1995

Acute and sublethal toxicity of seepage waters from garbage dumps to permanent cell lines and primary cultures of hepatocytes from rainbow trout (Oncorhynchus mykiss): a novel approach to environmental risk assessment for chemicals and chemical mixtures.

In order to evaluate the suitability of cytopathological criteria in isolated fish hepatocytes as endpoints in (eco)toxicological research, liver cells isolated from rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were exposed in vitro for up to 5 days to sublethal dilutions of two seepage water samples collected from garbage dumps. Hepatocytes were analysed with respect to acute (lactate dehydrogenase leakage) and sublethal toxicity (electron microscopy, stereology). In addition, acute toxicity (24 h) was tested in the piscine fibrocytic cell line R1 by means of crystal violet staining and neutral red retention. Acute toxicity in R1 cells and isolated hepatocytes could only be documented for sample I at dilutions of 1:2 and 1:4. This difference in toxicity could be corroborated by cytological alterations in isolated hepatocytes, which could be documented for dilutions of 1:100 and 1:8 in samples I and II, respectively. Ultrastructural changes were time- and dose-dependent and included reduction of hepatocellular volume, disturbance of intracellular compartmentation, modified heterochromatin distribution, transformation of rough endoplasmic reticulum into concentric membrane whorls, proliferation of lysosomes and cytoplasmic vacuoles, as well as reduction of hepatocellular glycogen. Although several hepatocellular reactions were found after exposure to either sample, the syndrome of ultrastructural alterations allowed clear differentiation between the two samples. Results illustrate that cytological effects far below macroscopically detectable damage can be discovered not only in intact fish, but also in fish cell culture systems. On the basis of the data presented, a multi-tiered test procedure for aquatic toxicity assessment exclusively based on tests with fish cell culture systems is proposed: (1) rapid screening for acute toxicity with permanent cell lines; (2) short-term tests with more complex, yet more sensitive systems such as primary hepatocytes with straightforward biochemical endpoints; (3) prolonged exposure of isolated hepatocytes in combination with ultrastructural and biochemical investigations as a sensitive tool to detect adverse effects at environmentally relevant toxicant concentrations.

Animals↗
PubMed1993

Cell cultures in the biocompatibility study of synthetic materials.

In vitro cytotoxicity (Neutral Red uptake, Kenacid Blue and MTT) and cytocompatibility (cell adhesion and proliferation) tests were applied to the biocompatibility study of a series of poly(ester-ether-ester) block copolymers of potential interest as biomaterials. Our results indicate that the copolymer extracts after 72 hours incubation with a 3T3 mouse fibroblast cell line do not induce significant toxic effects. Furthermore, human umbilical vein endothelial cells seeded on thin copolymer films show a normal pattern of growth. We conclude that the in vitro tests used are a valid instrument to evaluate the potential toxic action of synthetic materials on different cell compartments and that the tested materials seem to be promising for future applications in the field of biomedical devices.

3T3 Cells↗
PubMed1994

A study on reference standard for cytotoxicity assay of biomaterials.

The objective of the present study was to find a standard substance for use as a reference in the cytotoxicity assay of biomaterials, as an alternative to animal experiments in recent years. Eight kinds of rubber were made in a plate shape to keep their surface area at 1 cm2 against 10 ml of extract volume. They were extracted by the following three extraction methods (a) dynamic extraction at 200 rpm gyration on alumina balls at 37 degrees C for 24 h; (b) static extraction at 37 degrees C for 24 h and (c) extraction by heating in an autoclave at 121 degrees C for 60 min. At the end of each period each extract was examined for cell viability based on an evaluation by neutral red uptake. These methods were repeated up to seven times. Two kinds of chemicals were also tested. The extracts obtained were used to treat human gingival fibroblasts that have been cultured with DMEM supplemented with 5% fetal bovine serum into a 96 well tissue culture plate by 1 x 10(5) cells/ml, in an incubator aerated with 5% CO2, and 95% humidified air at 37 degrees C for 48 h. The extracts of ethylene-propylene, butyl, nitrile rubbers, and two kinds of chemicals yielded strong cytotoxicity in all three kinds of extraction methods, while chloroprene, fluorine-contained, isoprene, India, and silicone rubbers showed little cytotoxicity. The results obtained by the three kinds of extraction methods revealed no differences in the order of cytotoxicity of the materials tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Testing Alternatives↗
PubMedDate not supplied

Effect of dipyridamole, 5'-(N-ethyl)-carboxamidoadenosine and 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine on LOVO cell growth and morphology.

The effects of stable compounds acting on adenosine receptors, 5'-(N-ethyl)-carboxamidoadenosine (NECA: A2 and A1 adenosine receptor agonist) and 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX: selective A1 adenosine receptor antagonist) were evaluated in vitro on doxorubicin-resistant LoVo (LoVo-R) and doxorubicin-sensitive LoVo (LoVo-S) human metastatic cell lines by using the neutral red test for cell growth. The effect of dipyridamole, an adenosine uptake inhibitor, was also evaluated. The drugs had an inhibitory effect on LoVo cell growth. The association of the drugs with doxorubicin enhanced the inhibition of cell growth, particularly for NECA and PACPX on LoVo-R cells. Morphological observation with scanning electron microscopy indicated cytotoxicity of the tested compounds, alone or in association with doxorubicin both in LoVo-R or LoVo-S cells, supporting the hypothesis of inhibitory effect on tumor cell growth.

Adenosine↗
PubMed1993

Involvement of transforming growth factor-alpha and its receptor in the growth response of cultured human epidermal cells to retinoic acid.

Topical application of all-trans-retinoic acid (RA) to normal or photodamaged human skin causes pronounced growth effects on the epidermis (hyperplasia) by mechanisms poorly understood. This paper describes investigations concerning the possible involvement of transforming growth factor-alpha (TGF alpha) and its receptor in the growth stimulatory response of cultured human epidermal cells to RA. In a defined medium containing epidermal growth factor (EGF), RA (0.1-1.0 microM) was found to increase epidermal cell number and size as determined by uptake of neutral red dye. The stimulatory effect of RA was inhibited by a blocking monoclonal antibody directed against the binding domain of the EGF receptor. Relative to conditioned medium from cells grown in the absence of RA, conditioned medium from epidermal cells treated with RA contained increased concentrations of functionally active TGF alpha as determined by radioimmunoassays and radioreceptor assays. In addition, we found a marked reduction in the ability of TGF alpha to bind epidermal cells treated with RA. Decreased TGF alpha binding capacity is thought to be due to the ability of RA to stimulate TGF alpha, thereby resulting in EGF receptor binding and internalization. These results lead us to speculate that RA is capable of stimulating the secretion of TGF alpha by epidermal cells in vivo. TGF alpha may then act in an autocrine manner to mediate the hyperplastic response of the epidermis to topical RA treatment.

Antibodies, Monoclonal↗
PubMed1993

New experimental model system to study central regulation of intraocular pressure.

To elucidate the mechanisms of intraocular pressure (IOP) regulation by the central nervous system (CNS), the change in IOP following microiontophoretic application of oxytocin (OXT) onto the cat hypothalamus was investigated. Under sodium pentobarbital anesthesia, the experimental cat was fixed in a stereotaxic apparatus, immobilized and ventilated artificially through a tracheal cannula. The intraocular and blood pressures were continuously recorded using an electromanometer. A 3- to 5-barreled glass microelectrode was used; OXT and, as a control, saline were applied iontophoretically onto the hypothalamus according to the stereotaxic coordinates. On completion of the experiment, the animal was deeply anesthetized and perfused transcardially. Frozen sections of the brain were cut and stained with neutral red, and the tracks of the electrodes were reconstructed. The IOP was elevated gradually when OXT was applied onto the supraoptic nucleus. The latency and the amplitude of this response were 171 +/- 25 seconds and 0.99 +/- 0.46 mmHg (mean +/- SD; N = 4), respectively. The IOP showed no change after saline application to the same site. It was concluded that OXT-sensitive cells in the cat hypothalamus may play a role in regulating IOP. The combination of iontophoretic application of peptide and simultaneous IOP-monitoring as presented in this study provides a useful model system to study the mechanisms of IOP regulation by the CNS.

Animals↗
PubMed1993

[New experimental model of central regulation of intraocular pressure].

We investigated the effect on the intraocular pressure (IOP) of microiontophoretic injection of oxytocin (OXT) into the cat hypothalamus, to elucidate the mechanisms of IOP regulation by the central nervous system (CNS). Under sodium pentobarbital anesthesia, the cat was fixed in a stereotaxic apparatus, immobilized, and ventilated artificially through a tracheal cannula. The IOP and blood pressure were continuously recorded with an electromanometer. A 3- to 5-barreled glass microelectrode was used; OXT and saline as a control were injected iontophoretically into the hypothalamus according to the stereotaxic coordinates. On completion of the experiment, the animal was deeply anesthetized and perfused transcardially. Frozen sections were cut and stained with neutral red, and the tracks of the electrodes were reconstructed. The IOP was elevated gradually without change in blood pressure, when OXT was injected into the supraoptic nucleus. The latency and the amplitude of this response were 171 +/- 25 sec and 0.99 +/- 0.46 mmHg (average +/- SD; n = 4), respectively. The IOP showed no change after saline application to the same site. We concluded that OXT-sensitive cells in the cat hypothalamus may play a role in regulating IOP. This model may be useful to study the mechanisms of IOP regulation by the CNS.

Animals↗
PubMed1993

Intrinsic properties of the nonpeptide angiotensin II antagonist losartan in glomeruli and mesangial cells at high concentrations.

Intrinsic activities of the nonpeptide angiotensin II antagonist losartan were examined in a number of in vitro assays. Losartan produced contraction of rat isolated glomeruli at 100 mumol/l and of human mesangial cells at 1 to 100 mumol/l. Cell surface reduction was associated with disorganization of the alpha actin microfilament bundles. Losartan also stimulated cytosolic calcium concentration in cultured human mesangial cells at high concentrations (10-100 mumol/l). Losartan-dependent cytosolic free calcium concentration increase was not affected by nicardipine or 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride, whereas it was abolished in a calcium-free medium. There was a marked homologous desensitization response to losartan which was also obtained after pretreatment by EXP 3174 (2-n-butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl) biphenyl-4-yl)methyl]imidazole-5-carboxylic acid), the metabolite of losartan. The search for other agonistic effects of losartan in human mesangial cells including inositoltriphosphate formation, prostaglandin E2 production, [3H]leucine or [3H]thymidine incorporation was negative. Losartan and EXP 3174 were not toxic for human mesangial cells at the concentrations studied as judged by the absence of release of lactate dehydrogenase and the normal uptake of neutral red. These studies demonstrate that losartan exhibits glomerular effects in vitro only at high concentrations. Their relevance to in vivo situations is still questionable.

Angiotensin II↗
PubMed1995

Stimulation of antioxidative enzymes by paraquat in cultured Vero cells.

Different cellular and biochemical cytotoxicity indicators have been assessed to evaluate the damages caused in Vero monkey kidney fibroblasts after 24 h exposure to paraquat (PQ), a widely used bipyridyl herbicide highly toxic through the active oxygen species that it generates by redox cycling. Cell viability, estimated by the relative neutral red uptake (EC50 = 0.5 mM), was more sensitive to PQ than cell proliferation, measured by total protein content (EC50 = 5 mM). Cell growth was more extensively inhibited in the presence of fetal bovine serum than in its absence. PQ exposure was paralleled with higher intracellular specific activities of lactate dehydrogenase and phosphofructokinase, directly assayed in the 96-wells culture plates, whereas those of succinate dehydrogenase raised only 1.35-fold and hexosaminidase was almost unaltered by PQ. The intracellular specific activities of several antioxidative enzymes were also directly determined in the microtiter plates. At the highest PQ concentration used (10 mM) glutathione reductase activity increased 4-fold, while superoxide dismutase and glucose-6-P dehydrogenase activities increased 2- and 1.8-fold compared to untreated control cells. An 1.9-fold raise in glutathione-S-transferase activity was also observed in exposed cells. The results show the action in Vero cells of a complex regulatory defensive network against PQ-induced damages.

Animals↗
PubMed1995

[Detection of Clostridium perfringens toxins on cell cultures].

An important prerequisite for the possible application of cell culture systems as an in vitro method for the potency test of C. perfringens vaccines is the detection of cytotoxic effects of non-neutralized toxins on cell cultures. For this purpose eight cell lines were investigated in terms of the sensitivity to toxins using microscopy and the MTT assay. The results suggest that the MDCK cell line is a sensitive and specific detection system for epsilon-toxin. The VERO cell line is a suitable indicator for the detection of beta-toxin. Another task was the selection of a sensitive method for the analysis of cytotoxic effects of the toxins used. The neutral red and the tetrazolium-(MTT)-assays were compared for three cell lines (MDCK, FBTR, MA 104). The MTT assay was proved to be the optimal test system under our conditions.

Animals↗
PubMed1996

[Expression of the MDR1 gene in five human cell lines of medullary thyroid cancer and reversion of the resistance to doxorubicine by ciclosporin A and verapamil].

Medullary thyroid carcinoma (MTC) is frequently resistant to chemotherapy. Multidrug resistance (MDR) is one of the involved mechanisms. In this work we have studied the MDR1 gene expression in five MTC human cell lines that we have isolated and we have compared this expression to that of normal thyroid tissue. We have also tried to reverse the resistance to doxorubicin with verapamil (VRP) and ciclosporin A (CSA). MDR1 ARNm expression was studied and quantified by polymerase chain reaction (PCR) in normal and pathological thyroid tissues. The doxorubicin-induced cytotoxicity was evaluated with the 3,-4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) test, the neutral red (NR) uptake and with total glutathione (GSH) or intracellular lactate dehydrogenase (LDH) measurements. We found an increase of MDR1 ARNm in MTC as compared with normal tissues. Doxorubicin was cytotoxic after a 48-h coincubation with the cells. Three microM CSA and 10 microM VRP reversed the doxorubicin resistance only after a 48-h coincubation, generally followed with a 24 h-post-incubation. In these conditions, the GSH levels were decreased only by VRP in all the five cell lines. In conclusion, a chemoresistance related to the MDR1 gene overexpression was found in the five human MTC lines tested. VRP and CSA reversed the resistance to doxorubicin in all the MTC cell lines tested.

Antineoplastic Agents↗
PubMed1995

[The bioenergetic, cytochemical, and staining properties of normal hepatocytes and under functional loads, adaptations to environmental changes and in nonspecific injuries].

Two groups of cells were found among mouse liver cells and among hepatocytes in culture after their fixation and staining: weakly stained cells ("light" cells-LC), and strongly stained cells ("dark" cells- DC), the latter making about 30% of population. DC and LC do not differ in their capacity for DNA, RNA and protein synthesis, in contents of proteins, carbohydrates, lipids etc., and also in their ultrastructure. DC differ from LC in lower acidity (pH 6.5) of live cell cytoplasm, and in higher level of mitochondrial fluorescence after staining with Rhodamin 123. The live culture hepatocytes were seen in two alternative states: DC are able to become LC, and vice versa. The amount of DC increases after a high functional cell activation (partial hepatectomy, phlebotomy and hormonal stimulation), and after cell adaptation to modification of cultural medium conditions (modification of pH, hypo- and hypertonia, hyperthermia). In these cases hepatocytes maintain the capacity to granule formation after vital staining with Neutral red. After nonspecific moderate alteration (6% ethanol) of cultured hepatocytes, the acidity of cytoplasm and a temporary elevation of mitochondrial fluorescence may occur, in addition to the increase in the total number of DC. It is suggested that such a cell activation, both in normal physiological conditions and in the case of cell adaptation to modified cultural media, represents a general stress reaction of the cell preceding the "preparanecrosis" state.

Adaptation, Physiological↗
PubMed1996

Transplantation of encapsulated neonatal porcine islet-like cluster cells into diabetic rats.

OBJECTIVE: To evaluate the function of alginate poly-L-ysine membrane in protecting porcine to rat islet xenografts. MATERIAL AND METHODS: Newborn porcine islet-like cluster cells (ICC) were obtained by digesting pancreas with type XI collagenase. After 2-3 days' incubation, encapsulated ICC were formed through instrument. In vitro, insulin release and insulin stimulating test was performed. In vivo, encapsulated ICC were transplanted into streptozotocin-induced diabetic rats. RESULTS: Unlike ICC only (insulin can be tested within 14 days) encapsulated ICC maintained insulin release at certain level for nearly 28 days. High insulin response to 16.7 mM glucose and 10 mM theophylline significantly differed from low insulin response to 5.6 mM or 16.7 mM glucose (P < 0.01). Extended survival of porcine ICC xenografts implanted in diabetic rats had been achieved by encapsulation. After transplantation, rats sustained normal glycemia for average 4 months. The capsules with ICC obtained from experimental group could be stained with neutral red. Insulin release of survival capsules with ICC in response to 16.7 mM glucose and 10 mM theophylline was 3 times more as compared to the insulin response to 5.6 mM glucose. Sections of encapsulated ICC (H&E) showed fairly good cell clusters and the membranes were generally free of fibrosis and host cell adherence. CONCLUSION: Artificial membrane can protect discordant islet xenografts from rejection.

Animals↗
PubMed1996

Characterization of in vivo gene transfer into the arterial wall mediated by the Sendai virus (hemagglutinating virus of Japan) liposomes: an effective tool for the in vivo study of arterial diseases.

Recently, much attention has been paid to the arterial gene transfer technique using several vector systems. In the present study, we experimentally examined the transfection efficiency of the exogenous gene, the duration of gene expression, and the cytotoxic effect of the hemagglutinating virus of Japan (HVJ) liposome on the intact arterial wall to evaluate its effectiveness for the study of arterial diseases. To evaluate the transfection efficiency and duration of gene expression, pSV beta-galactosidase was transferred into the carotid arterial wall of rabbits. The cytotoxic effect of HVJ liposomes on the vascular cell components was also evaluated by a neutral red assay in vitro and scanning electron microscopy in vivo. HVJ liposomes could achieve highly efficient gene transfection into the medial smooth muscle cells of intact arteries at 150 and 760 mmHg of pressure (mean = 85.3% and 93.5% of total smooth muscle cells, respectively) without any inflammatory reaction. The introduced exogenous gene was expressed for at least 14 days. In addition, the cytotoxicity for the arterial smooth muscle cells and endothelial cells induced by HVJ liposome vehicles at routinely used concentrations (5,000 to 10,000 hemagglutinating activity units/ml) was minimal both in vitro and in vivo. As an example, we introduced human vascular endothelial growth factor cDNA, which was driven with cytomegalovirus enhancer and beta-actin promoter, into the rabbit carotid arteries, and it induced not only angiomatoid proliferation of endothelial cells forming irregular vascular channels but also intimal hyperplasia. Based on these findings, we conclude that HVJ liposome-mediated arterial gene transfer is a highly efficient, noninvasive, and effective gene delivery method for the study of vascular disorders.

Animals↗
PubMed1996

Cytotoxicity, pH, and glucose degradation products in four different brands of PD fluid.

This study was undertaken in order to evaluate differences in pH, cytotoxicity, and concentration of glucose degradation products between different peritoneal dialysis (PD) fluids. Four brands of commercial PD fluids were selected: Gambrosol, Bieffe, Dianeal, and Lock-olys, with a glucose concentration between 3.86% and 4.25%. The cytotoxicity of unused PD fluid was measured as inhibition of cell growth after three days' exposure of a fibroblast cell line using neutral red vital stain. The concentration of 2-furaldehyde, 5-hydroxymethyl-furaldehyde (5-HMF), acetaldehyde, formaldehyde, glyoxal, and methylglyoxal was analyzed by high-performance liquid chromatography technique. All the investigated PD fluids were cytotoxic and contained noticeable amounts of aldehydes. No major differences were observed in pH, although one fluid with a slightly lower pH was found to be less cytotoxic. This fluid also contained less formaldehyde, acetaldehyde, methylglyoxal, and glyoxal but instead contained more 5-HMF. We conclude that all brands of PD fluid are cytotoxic due to glucose degradation products and that they contain noticeable concentrations of low-molecular aldehydes. Our results also demonstrate that the concentration of 5-HMF, used as a regulatory control of PD fluids, does not correlate well with cytotoxicity.

Acid-Base Equilibrium↗