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[Cytotoxic effects of restorative materials on early passage cultured cells derived from human gingiva (in vitro)].

This study evaluated the cytotoxic effects of three common restorative materials on early passage cultured cells derived from human gingiva (HG cells). Other early passage cultured cells derived from human dental pulp tissues (HP cells) and L cells established obtained from mouse subcutaneous tissues, were also examined. Cytotoxicity was assessed according to the uptake of neutral red and dye exclusion with nigrosin. The cytotoxicity was in the order of glass ionomer cement (highest), light cured composite resin and dental amalgam. HG cells were the least sensitive to the materials tested. Both filtration of extracts and addition of serum into the extract affected the cytotoxicity. Glass ionomer cement increased the acidity of the extract, but the two other materials did not. Dissolution of metal ions, aluminium, tin, copper, mercury and zinc occurred from the materials. The extract of the composite resin had a different absorbance. This study disclosed differences in the cellular reaction to the restorative materials. Thus, utilization of early passage cultured cells is necessary for biological evaluation of the dental materials in vitro.

Animals↗

[The vestibular nuclei of the guinea pig: their structural and topical organization].

Topographical and cytoarchitectonical organization of the vestibular nuclear complex of guinea pig has been studied. Four vestibular nuclei: superior, lateral, medial, inferior as well as cellular groups (I, F, Y, X, Z) associated with the vestibular nuclei were described on the basis of neutral red stained preparations. The precise delimitation and the stereotaxic coordinates of the vestibular nuclei were performed by means of the analysis of 50 microns thick frontal serial slices treated by glycerine.

Animals↗

Intrinsic and extrinsic factors influencing properties and growth patterns of identified leech neurons in culture.

An analysis has been made of intrinsic mechanisms influencing growth patterns of 2 identified leech interneurons in tissue culture. These small cells (known as DL and VL) display unusual arborizations in the ganglion. The distinctive branching patterns resemble the letter "T" for VL and the letter "Y" for DL. DL and VL cells contain serotonin and can be identified in situ with Neutral red. 1. DL and VL cells were isolated and cultured on 2 homogeneous substrates, concanavalin A (Con A) and leech extracellular matrix extract (ECM). Individual DL and VL cells in culture retained their ability to fire in spontaneous, rhythmical bursts. The synaptic connections formed in culture were specific and differed from those made by other serotonergic neurons. 2. On both Con A and ECM the neurons sprouted to reproduce their characteristic patterns. Every DL or VL cell that grew processes on ECM developed the appropriate Y- or T-shaped pattern. On Con A, most but not all of the cells showed specific Y or T patterns. The probability of sprouting was higher on Con A than on ECM. 3. Disruption of microtubular assemblies in freshly isolated DL and VL cells by treatment with nocodazole did not affect the later outgrowth of specific patterns in culture. 4. These results provide evidence that adult DL and VL leech neurons retain intrinsic information for determining a variety of their properties in culture: In particular, they reproduce type-specific neuritic patterns in the absence of extrinsic guidance cues. Moreover, the intrinsic pattern determining mechanism is not lost after the destruction of microtubular arrays within the cytoskeleton.

Animals↗

[Changes in the composition of the lysosomes in hepatocytes cultured in vitro in modelling pathological states].

The monolayer cultures of newborn rats hepatocytes were treated with substrate starvation (20 min.) anoxia accompanied with substrate starvation (1 h.), following rehabilitation (1 h.) and cooling (4 degrees C, 30 min). After the neutral red staining we have examined the alterations in lysosomes quantity and the development of phagocytosis; we also tested the hepatocytes distribution due to the amount of the second lysosomes per cell. We have shown that a short time pathological treatment caused a decrease in large lysosomes (1-3 microns) amount in significant portion of hepatocytes. There were neither increase in phagosomes amount per cell nor alterations in the quantity of cells, containing phagosomes. However a long time pathological treatment caused the increase in phagosomes and cells. We propose that initial stage of lysosomal apparatus alterations, connected with reduction of CAMP level, is accompanied by a stamping of large lysosomes.

Animals↗

[Action of high hydrostatic pressure on the fluorescence of respiratory metabolic components and on the staining capacity of the skeletal muscles in the frog].

The effect of high hydrostatic pressure (100-1400 at) on frog skeletal muscle was studied. Superthreshold pressures (above 600 at) accompanied by muscle contracture results in the intensification of sorption of both basic (neutral red) and acid (phenol red) dyes, and in the growth of the number of oxidated forms of respiratory metabolic components--flavoproteins and pyridine nucleotides under study. Subthreshold pressures reduce the tinctorial ability of muscles and decrease oxidative processes caused by the maintenance of muscle in small volume of the Ringer solution.

Absorption↗

Deproteinization of influenza virus in the presence of rimantadine.

Virion deproteinization and viral RNA transport to the isolated cell nuclei have been studied in the presence of rimantadine with rimantadine-sensitive influenza viruses fowl plague (H7N7), A/Krasnodar/101/59 (H2N2) and rimantadine-resistant influenza strains (WSN/H1N1 and A/Krasnodar/101/59-R). Rimantadine failed to affect deproteinization during incubation with the isolated cellular plasma membranes as well as the transport to isolated cell nuclei of the viral RNA of either sensitive or resistant strains of influenza virus. Using photosensitive viruses (labelled with neutral red) rimantadine exerted dissimilar effects on deproteinization of the sensitive and resistant influenza virus strains. The possible effects of rimantadine on influenza virus deproteinization is discussed.

Adamantane↗

A study of osteoclasts on calvaria of normal and osteopetrotic (mi/mi) mice by vital staining with acridine orange.

A novel staining procedure for enumerating osteoclasts on neonatal mouse calvaria with the vital fluorescent dye acridine orange is described. It has the advantage over Barnicot's neutral-red method in that the nuclei and cytoplasm of the osteoclast are stained differentially. The osteopetrotic calvarium (mi/mi) has fewer multinucleate osteoclasts than its normal counterpart (mi/+) and they are differently distributed. The osteopetrotic calvarium has more mononucleate cells which stain like osteoclasts with acridine orange than the normal calvarium and these cells also are differently distributed. These mononuclear cells may be mononuclear osteoclasts or their precursors. These observations suggest that the defect resulting in this osteopetrosis lies with osteoclast differentiation.

Acridine Orange↗

Photodynamic toxicity of hematoporphyrin derivatives to human keratinocytes in culture.

Human keratinocytes in culture were able to take up hematoporphyrin derivatives (HPDs) used during photodynamic chemotherapy of tumors. In the absence of light, HPDs showed no cytotoxic effects to keratinocytes. However, after irradiation with visible light, HPDs induced immediate cytotoxicity as measured by the neutral red uptake assay. On the other hand, cell attachment as measured by protein estimation was not affected. When the cells treated with HPDs and irradiated with light were cultured for a further 72 h, they partially lost their ability to attach to the collagen surface. Most of the cells remaining attached after 72 h were no longer viable following treatment with HPDs and light. All parameters measured depended on the intracellular concentration of HPDs used (7-50 ng/10(5) cells) and the time of irradiation (0-30 min). These results suggest that human keratinocytes are a good model to study cytotoxic effects of photodynamically active drugs. Further, keratinocytes were unable to recover after damage caused by HPDs and light.

Cells, Cultured↗

Multilayered keratinocyte culture used for in vitro toxicology.

Human keratinocytes are the most appropriate target cells for evaluating mechanisms of skin cytotoxicity and pharmacology of chemical agents. After having formed a confluent stratified epithelium with proliferating basal cells and differentiated cell layers, human keratinocytes were harvested after enzymatic detachment as stable, three-dimensional cell aggregates or used as adherent multilayers in microtiter plates to study the local cytotoxicity of different toxic compounds. The advantage of this test is that it uses the adequate target cells and that it evaluates both the ability of the test chemical to penetrate several cellular layers as well as the ability to interfere with cellular function. The end points are cell viability and cell metabolism, which are determined by neutral red uptake and MTT reduction, respectively. For the 13 chemicals evaluated in this study we found good correlation (r = .819) between the potency rankings of keratinocyte NR 50 values and in vivo irritancy data. There was also good agreement (r = .945) between ranking of these chemicals according to midpoint toxicity of both the 3T3 assay and the keratinocyte assay. This test system might be at the present stage a supplementation of the current test battery, which shall replace in vivo irritation tests like the Draize test.

Cells, Cultured↗

[Cytological examination of the amniotic fluid in normal pregnancy and in cases of fetal central nervous system abnormalities].

Cytological and biochemical investigations were carried out on 60 samples of amniotic fluid obtained from 30 pregnant women with risk for development of fetal central nervous abnormality (FCNA) and 30 pregnant women in whom prenatal diagnosis was indicated for other reasons (control group). Cytological evaluation was done in an interference-polarization Nomarski microscope evaluating the cells in direct preparation and after staining with neutral red. Parallelly with cytological evaluation alpha-1-fetoprotein (AFP) and acetylcholinesterase (AChE) were determined in amniotic fluid. In three cases with open neural tube anomaly characteristic cells with strongly puckered and vacuolized cytoplasmic membranes were found. In these cases the levels of AFP and AChE exceeded the normal range. In two cases of closed abnormalities of the central nervous system diagnosed by ultrasonography no abnormalities were note by cytological and biochemical methods. The study confirmed the usefulness of the cytological examination of amniotic fluid, as a method supplementing biochemical and ultrasonographic investigations as part of prenatal diagnosis.

Acetylcholinesterase↗

[Trichomonas vaginalis infection in pregnant women and newborn infants].

We studied 149 pregnant women at delivery and 113 newborns in order to determine the frequency of T vaginalis infection. Neutral red stain, dark field microscopy and culture methods were used with comparable yields. The age of women ranged from 12 to 43 years. A 27.5% infection rate was detected in mothers but all newborns were free from infection. Infection rates in relation to age among mothers confirmed previously reports.

Adolescent↗

[Cytotoxicity of dental filling materials in primary cultured cells derived from human dental pulp (in vitro)].

Primary cultured cells derived from human pulp tissue were used for examining the cytotoxicity of nine commercial filling materials used in restoration and root canal procedures. Cell growth was evaluated by photo pattern analysis, cell nuclei counting, and neutral red uptake. L-strain cells were also used for comparison. Primary cultured cells from either the coronary or radicular part of the pulp tissue were less sensitive to the tested materials, than were L-cells. As for sensitivities over time following mixing of the materials, the cytotoxicity of the three restorative materials was immediately moderate but disappeared 24 hours after mixing. The other six root canal filling materials yielded similar moderate to high cytotoxic levels immediately after mixing and the effects continued. The order of cytotoxicity was: Diaket and Calvital (highest); Pulp canal sealer, AH-26, and Canals (high); and then Kloroperka and three restorative materials (moderate). The differences in cellular reactions between primary and strain cells was considered to be due to cell origin, i.e. diploid or heteroploid. The importance of using primary cultured cells from designated target tissues when evaluating various materials in vitro was discussed.

Balsams↗

Quantitative aspects of cellular autophagocytosis. Morphometric and cell fractionation studies.

The mode of action of agents and treatments known to induce the appearance of autophagic vacuoles (AV) were studied in mouse liver, pancreatic and seminal vesicle cells by morphometric evaluation of the time-dependent changes in the volume fraction of AVs following suppression of sequestration by cycloheximide. Rapid decrease of the AV compartment was observed in cells of animals pretreated with agents including Triton X-100, pilocarpine, leupeptin and estron acetate. The half-life of AVs, estimated from the decay, range between 5.3 to 8.7 min, which does not deviates from normal steady state values. In contrast, no regression or very slow decay was seen in cells of animals pretreated with methylamine, chloroquine and vinblastine (VBL). We think that the main mechanisms responsible for the enlargement of the AV compartment in these experiments are: stimulation of the sequestration under the effect of agents which maintain the half-life of AVs within the physiologic range of 5-10 min and accumulation of AVs due to their prolonged lifetime under the effect of acidotropic agents and VBL. However, our data suggest than in addition to the accumulatory effect on AVs, stimulation of sequestration may also play a role in the enlargement of the AV compartment after treatment with VBL. While it was not possible to isolate a relatively pure fraction of early AVs from the liver, a light and a dense AV fraction was successfully purified in our laboratory from the pancreas treated with either VBL or neutral red. Preliminary enzymological and morphological evidence is presented that the light AVs are of autophagosomal, whereas the dense ones are of autolysosomal nature.

Animals↗

[In vitro and in vivo studies of RNAse of Bacillus intermedius].

Cytotoxicity of RNAase from Bacillus intermedius was studied in vitro and in vivo. It was shown that the enzyme had slightly pronounced cytotoxicity according to the tests with inhibition of cell proliferation and biosynthesis of cell nucleic acids. The RNAase was also shown to impair the vital staining by neutral red. The efficiency of the impairment much more depended on the enzyme catalytic activity than on the proliferation and biosynthesis of nucleic acids. In vivo toxicity of RNAase from B. intermedius was 3-5 times higher than that of pancreatic RNAase. Possible mechanisms of the different toxicity of the enzymes are discussed.

Amnion↗

Proliferative potential of degranulated murine peritoneal mast cells.

The fate of mast cells after degranulation was investigated. Purified peritoneal mast cells of WBB6F1-+/+ mice were sensitized with monoclonal anti-dinitrophenol (DNP) IgE antibodies and stimulated with DNP conjugated with human serum albumin. Mast cells were vitally stained with neutral red, and highly degranulated mast cells were identified under a phase-contrast microscope and individually picked up with the micromanipulator. When these highly degranulated mast cells were individually plated in methylcellulose, their potential to produce a cluster or a colony was comparable to that of morphologically intact mast cells. Moreover, when highly degranulated mast cells were injected into the skin of genetically mast cell-deficient WBB6F1-W/Wv mice, the proportion of injection sites at which mast cell clusters appeared was comparable to the value observed when morphologically intact mast cells were injected. The present result indicates that proliferative potential of mast cells is not reduced by their degranulation.

Animals↗

The role of somatostatin and serotonin in the beta-adrenoceptor regulation of gastric function. An experimental study in dogs.

CHAPTER 1. The gastric functions are regulated in a complex manner by the autonomic nervous system, sympathetic and parasympathetic, and hormones and moreover by a system of peptide-containing cells and nerves. The sympathetic influence is mediated by alpha- and beta-adrenoceptors, beta-adrenoceptor agonists in general inhibit the gastric functions. The effects of the beta-adrenoceptors are probably mediated indirectly, and the responsible mechanism may be via release of and endogenous mediator--somatostatin or serotonin. The purpose of the present study, described in the survey, is to examine whether somatostatin or serotonin acts as a mediator for the gastric effects in vivo of beta-adrenoceptor agonists. The proposed mediator must present the following characteristics: A. Inhibitory effects of "physiological" doses in vivo. B. Inhibitory characteristics similar to those of the beta-adrenoceptor agonists. C. A beta-adrenoceptor mediated release. CHAPTER 2. In this chapter the known effects of beta-adrenoceptor agonists on gastric functions (acid secretion, pepsin secretion, antral motility and mucosal blood flow) are presented. The possible mechanisms mediating these effects are mentioned, and the effects of somatostatin and serotonin are reviewed. Beta-adrenoceptor agonists inhibit the acid secretion in vivo in several species, but stimulate in vitro. A similar pattern is probable as for the pepsin secretion, but sufficient results are not available in order to draw a certain conclusion. The antral motility is inhibited both in vivo and in vitro. The mucosal blood flow is increased compared to the acid secretion, however the results points to variable effects depending on the circumstances of the experiments. On the basis of the reviewed effects of somatostatin it is concluded that the effects are inhibitory, but the inhibitory characteristics are not known. A beta-adrenoceptor mediated release of somatostatin has been found, but it has not been examined selectively for the stomach. The gastric effects of serotonin has been sparsely examined and it is not possible to determine the effects as parts of a certain characteristic. It is concluded that neither somatostatin nor serotonin have been examined sufficiently to determine which is the proposed mediator. CHAPTER 3. The materials and methods used are reviewed. Dogs were used and various parameters were measured--acid secretion by titration, pepsin secretion by enzymatic process with haemoglobin as substrate, antral motility by registration of intraluminal pressure, mucosal blood flow by the clearance of neutral red, somatostatin by radioimmunoassay.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic beta-Agonists↗

Plaque enhancement effect of sodium thiosulfate for foot-and-mouth disease viruses.

A plaque enhancement effect by the addition of sodium thiosulfate for foot-and-mouth disease viruses was demonstrated when this salt was incorporated in agar and in agarose overlay media. Most of the mechanism is obscure, however, as one of the effects is that sodium thiosulfate seems to interact in a reversible manner against the plaque inhibitor action of neutral red in cellular cytoplasm. A plaque inhibitor contained in agar could be removed in some degree by the addition of this salt.

Agar↗

Mediating role of metabolic activation in in vitro cytotoxicity assays.

Enzymatic activation of polycyclic aromatic hydrocarbons (PAHs) and its effect on cytotoxicity were studied using the neutral red viability assay as the end point. Benzo[a]pyrene was progressively cytotoxic to human hepatoma (HepG2) cells over a 1- to 3-d period, and after induction of monooxygenase activity with a polychlorobiphenyl (PCB) mixture (Arochlor 1254), cytotoxicity was increased about threefold. Concomitant with Arochlor exposure was an increase in the activity of 7-ethoxycoumarin odeethylase, which could be inhibited by exposure to alpha-naphthoflavone. Human keratinocytes (NHEK), but not fibroblasts (HFF), were sensitive to the cytotoxicity of benzo[a]pyrene. However, preexposure of the keratinocytes to Arochlor did not increase their sensitivity to benzo[a]pyrene. Neither the keratinocytes, fibroblasts, nor HepG2 cells were sensitive to acenaphthene. Addition of hamster or rat hepatic S9 mix, however, resulted in toxicity from benzo[a]pyrene and acenaphthene. 7,12-Dimethylbenz[a]anthracene was only mildly cytotoxic to the fibroblasts, and its cytotoxicity was not enhanced in the presence of rat or hamster S9 mix. Exposure of the HepG2 cells to 7,12-dimethylbenz[a]anthracene showed progressive toxicity over a 1- to 3-d period. Prior exposure of the HepG2 cells to Arochlor did not enhance their sensitivity to 7,12-dimethylbenz[a]anthracene. Human keratinocytes were sensitive to 7,12-dimethylbenz[a]anthracene, with cytotoxicity markedly increasing over a 1- to 3-d period.

9,10-Dimethyl-1,2-benzanthracene↗