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PubMed1999

Cytotoxicity tests of in situ polymerized resins: methodological comparisons and introduction of a tissue culture insert as a testing device.

The in vitro cytotoxic potential of six commonly used methacrylate polymers was evaluated using human oral fibroblast cultures with different cell-material contact systems. A tissue culture insert was introduced to test resin-released components. Both acute and delayed cytotoxic effects of resin were quantified by cellular enzymatic and DNA synthetic activities of fibroblasts over a 6-day exposure period. Resin toxicity was material-dependent. Statistical analysis showed that the experimental conditions significantly contributed to the overall toxicity and the cytotoxicity pattern for a given material. DNA synthesis activity of human oral fibroblasts assayed by 3H-thymidine incorporation was more sensitive to resins than cellular enzyme activity, as determined by tetrazolium bromide reduction. However, extended exposure increased the cytotoxicity of all resins, as measured by tetrazolium bromide reduction, which seemed to be a better indicator of the development of resin toxicity than 3H-thymidine incorporation. Removal of the oxygen inhibition layer on resin specimens partially enhanced cell viability, indicating that this surface layer together with other unknown factors contributed to resin toxicity.

Cell Survival↗
PubMed1992

Differential cholinergic innervation within functional subdivisions of the human cerebral cortex: a choline acetyltransferase study.

The distribution of cholinergic fibers in the human brain was investigated with choline acetyltransferase immunocytochemistry in 35 cytoarchitectonic subdivisions of the cerebral cortex. All cortical areas and all cell layers contained cholinergic axons. These fibers displayed numerous varicosities and, on occasion, complex preterminal profiles arranged in the form of dense clusters. The density of cholinergic axons tended to be higher in the more superficial layers of the cerebral cortex. Several distinct patterns of lamination were identified. There were also major differences in the overall density of cholinergic axons from one cytoarchitectonic area to another. The cholinergic innervation of primary sensory, unimodal, and heteromodal association areas was lighter than that of paralimbic and limbic areas. Within unimodal association areas, the density of cholinergic axons and varicosities was significantly lower in the upstream (parasensory) sectors than in the downstream sectors. Within paralimbic regions, the non-isocortical sectors had a higher density of cholinergic innervation than the isocortical sectors. The highest density of cholinergic axons was encountered in core limbic structures such as the hippocampus and amygdala. These observations show that the cholinergic innervation of the human cerebral cortex displays regional variations that closely follow the organization of information processing systems.

Adult↗
PubMed1976

Transformation of cells by rous sarcoma virus: cytoplasmic vacuolization.

Chick embryo cells transformed by the Bryan "high titer" strain of Rous sarcoma virus (RSV-BH) are heavily vacuolated. A variety of microscopic techniques have been used demonstrating that the vacuoles are cytoplasmic, bounded by membrane, and are composed largely of water. Proteins, lipids, glycoproteins, glycolipids, glycosaminoglycans, glycogen, and nucleic acids were undetectable in the vacuoles. Physiological requirements for development of the vacuoles, and reversal of vacuolization, were examined in cells infected with a virus mutant, RSV-BH-Ta, which induces reversible temperature-dependent transformation. Na+ was the only component of the cell culture medium found essential for both the development and reversal of vacuoles. Glucose depletion or dinitrophenol treatment inhibited vacuolization, suggesting a possible energy requirement in the vacuolization process. Ouabain, an inhibitor of Na+-K+ ATPase, enhanced vacuolization, but a variety of other substances affecting cell surface components were in active. Two sugars, glucosamine and mannosamine, prevented the disappearance of vacuoles. The observations suggest that cellular vacuolization may be a normal physiological response to an increase in water and Na+, and, in the specific case of transformation by RSV-BH, may be relevant to the physiological basis for malignancy.

Adenine↗
PubMed1978

Synthesis of as-triazines as potential antiviral agents.

Four acenaphtho[1,2-e]-as-triazines and 11 5,6-diaryl-as-triazines, all substituted with an aliphatic or aromatic amino function in the 3-position, were synthesized. Two acenaphthotriazines were active against vesicular stomatitis virus in tissue culture.

Animals↗
PubMed1994

Effects of hypothermia on the survival and cryopreservation of minipig ileal cells and Chinese hamster ovary cells.

Temperature of culture can be used to modulate cellular metabolism for improving small intestinal cell culture and cryopreservation. An hypothermia pretreatment (2 days at 25 degrees C and 3 hours recovery at 37 degrees C) improved hamster cell survival to freeze-thaw damage (p < 0.01) but decreased the survival of 2 immortal pig ileal cell lines even though epithelioid IPI-2I cells were more tolerant to hypothermia than IPI-1 fibroblasts. Epithelioid cells survived 3 days at 25 degrees C with unaltered expression of cytokeratin-18 whereas colonies of fibroblasts did not survive more than a day at 25 degrees C (p < 0.001). These results suggest that hypothermia-tolerance of pig ileal cell lines might differ according to cell lineage calling for further experiments on small intestinal primary cell culture.

Animals↗
PubMed2002

Modulation of PGE(2) and TNFalpha by nitric oxide and LPS-activated RAW 264.7 cells.

Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNFalpha play major roles in inflammation and they are synthesised mainly by macrophages. Their syntheses have been shown to be regulated by several factors, including nitric oxide, a further important macrophage product. Since both positive and negative regulations of PGs and TNFalpha synthesis by NO have been reported, we sought to understand the mechanisms underlying these opposite NO effects by using a recent class of NO releasing compounds, the NONOates, which have been shown to release NO in a controlled fashion. To this aim, we analysed the effect of NO released from PAPA/NO (t1/2 15 min) and DETA/NO (t1/2 20 h) in RAW 264.7 cells. Both NONOates were used at the same concentrations allowing the cell cultures to be exposed either at high levels of NO for brief time (PAPA/NO) or at low levels of NO for long time (DETA/NO). We found that the two NONOates had opposite effect on basal TNFalpha release, being increased by PAPA/NO and decreased by DETA/NO, while they did not affect the release stimulated by LPS. At variance, both NONOates increased the basal PGE(2) production, while the LPS-stimulated production was slightly increased only by PAPA/NO. The modulation of PGE(2) synthesis was the result of the distinct effects of the two NO-donors on either arachidonic acid (AA) release or cyclooxygense-2 (COX-2) expression, the precursor and synthetic enzyme of PGs, respectively. Indeed, in resting cultures AA release was enhanced only by PAPA/NO whereas COX-2 expression was moderately upregulated by both donors. In LPS activated cells, both NONOates induced AA release, although with different kinetics and potencies, but only DETA/NO significantly increased COX-2 expression. In conclusion, by comparing the activities of these two NONOates, our observations indicate that level and time of exposure to NO are both crucial in determining the molecular target and the final result of the interactions between NO and inflammatory molecules.

Animals↗
PubMed2000

Functional and ultrastructural cell pathology induced by fuel oil in cultured dolphin renal cells.

Investigations were undertaken to elucidate in a marine mammal renal cell culture system the toxicity and some of the mechanisms of cytopathology in a standardized preparation following exposure to No. 1 fuel oil. Cell survivability of a cultured SP1K renal cell line from the Atlantic spotted dolphin Stenella plagiodon was reduced in a dose-dependent manner after a 12-h exposure to fuel oil. Early morphologic changes reflecting cytotoxicity, as revealed by transmission electron microscopy, included enlarged rough endoplasmic reticula, cytoplasmic vacuolization, and degenerative cytoplasmic inclusions, but mitochondria remained resistant. Assessment of extracellular proton loss by microphysiometry of cultured cells revealed fuel oil-induced enhancement of proton loss that was dependent upon both protein kinase C and renal epithelial Na(+)/H(+) counter-transport functioning, as the specific inhibitors H-7 and amiloride reduced this stimulatory petroleum effect. Cell cycle progression and apoptosis (programmed cell death) were studied in dolphin renal cells exposed to fuel oil for 12, 24, and 48 hours. The toxicant increased the percentage of cells in GO/GI phase and decreased the percentage of cells in S phase starting after 24 hours. The number of cells undergoing early apoptosis was also increased after 24 hours.

Animals↗
PubMed1995

Development and intralaboratory evaluation of an in vitro human cell-based test to aid ocular irritancy assessments.

A human cell-based in vitro method was developed to screen for ocular irritancy potential of aqueous compatible and incompatible test agents, such as liquids, insoluble solids, powders, granulars, emulsions, and acids/alkalis. Methods were developed for topical application (an exposure that mimics in vivo testing) and wash-off of test substances on the epithelial surface of human skin derived epithelial-fibroblast cocultures (Skin2 Model ZK1200 from Advanced Tissue Sciences). These cultures contain noncornified stratified squamous epithelium, providing a three-dimensional in vitro model that resembles noncornified mucosal epithelium, such as cornea and conjunctiva. The hypothesis tested and confirmed in this work was that the rate of cytotoxicity induced by topical application of test substances to the stratified epithelial cell cultures would correlate with ocular irritancy. Test substances were applied to the cell system for up to 30 min, and cytotoxicity was measured as decreased 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) vital dye metabolism. The time (in minutes) of exposure to test agent that reduced MTT metabolism to 50% of control levels (the t50 value) was calculated for each test substance, and these values showed a good correlation (r = 0.87) with historic rabbit low-volume eye test maximum average scores (MAS) for a range of liquid, solid, granular, powder, and other test materials, including consumer product formulations and ingredient chemicals. Additionally, t50 values confirmed the mild nature of selected cosmetic formulations. Taken together, these data indicate that this in vitro test, termed the tissue equivalent assay, is a valuable tool to screen for irritancy of test substances to mucosal tissues, such as cornea and conjunctiva.

Animal Testing Alternatives↗
PubMed1997

Tetracycline-induced steatosis in primary canine hepatocyte cultures.

Primary hepatocyte cultures prepared from male beagle dog liver were used to determine susceptibility of the canine liver to tetracycline-induced steatosis. The effects of the drug on mitochondrial lipid metabolism and intracellular triglyceride accumulation were monitored at the same time that steatosis was detected by light microscopy and quantitated using lipid-specific stains. Exposure of primary canine hepatocyte cultures to tetracycline for 24-48 h resulted in concentration-dependent, significant increases in the Oil Red O-stained lipid inclusions. Microscopic examination of the total stained areas suggested that increases over control levels were due primarily to the increase in the size of the lipid inclusions rather than in the number. Biochemical analyses for triglyceride content and histological staining with Nile red, another neutral lipid-specific dye, confirmed a specific increase in intracellular triglyceride following a 24-h exposure to noncytotoxic levels of tetracycline beta-oxidation studies based on the oxidation of [14C]palmitic acid or [14C]palmitoyl carnitine demonstrated a concentration-dependent inhibition of mitochondrial but not peroxisomal beta-oxidation in hepatocytes after a 24-h exposure to tetracycline. In vitro incubation of tetracycline with mitochondria isolated from dog liver showed similar concentration-dependent inhibition. This study clearly indicates that the canine hepatocyte is susceptible to tetracycline-induced steatosis. Triglyceride accumulation was concomitant with the inhibition of mitochondrial lipid metabolism, indicating that this is a primary mechanism leading to steatosis in dog hepatocytes following tetracycline exposure.

Animals↗
PubMed1983

Biogenic amines in the brain of the honeybee, Apis mellifera.

Fluorescence histochemistry with glyoxylic acid has been used in close conjunction with detailed anatomical studies (Mobbs 1982) to investigate the distribution of fluorogenic amines in the brain of the honeybee, Apis mellifera. In addition, the concentration and distribution of biogenic amines in the brain of the bee have been determined using highly sensitive radioenzymatic techniques and high performance liquid chromatography. The cerebral ganglia of the bee contain similar amounts of dopamine and serotonin, more dopamine than octopamine, and very low levels of noradrenaline. Slow fading green fluorescence, typical of catecholamines, was located throughout the cerebral ganglia, and was particularly intense in the central body and mushroom body neuropils. Results indicate that the fluorescence in the mushroom body neuropils is largely extrinsic in origin. Both dopamine and serotonin were detected in the calyces and alpha-lobe of the mushroom bodies, and in the antennal lobe. In the optic lobe, however, serotonin was found, but only low levels of dopamine were detected. Slow fading green fluorescence was replaced in the optic lobes by fluorescence which faded rapidly in the excitation light. The non-fluorogenic amine octopamine was found in the mushroom bodies and in the neuropils of the optic lobes, with the largest amounts of octopamine in the optic lobes associated with the neuropil of the medulla. The possibility that intrinsic neurones of the mushroom body neuropil are octopaminergic is discussed.

Animals↗
PubMed1994

Permeabilization of cells of hemopoietic origin by extracellular ATP4-: elimination of osteoclasts, macrophages, and their precursors from isolated bone cell populations and fetal bone rudiments.

Skeletal tissues contain, apart from cells of the osteogenic and chondrogenic lineage, cells of hemopoietic origin, e.g., macrophages, osteoclasts, and their precursors. In the present study we examined the sensitivity for extracellular ATP4- of the above-mentioned cell types in freshly isolated, bone-derived cell populations and in explanted fetal metatarsal bones. Cells of hemopoietic origin reacted to the presence of ATP4- with an increased permeability for impermeant cytotoxic molecules, e.g., ethidium bromide (EB), thiocyanate (KSCN), and an increased non-ion selective membrane conductance. As a consequence, these cells could be killed by a short treatment with adenosine-5' triphosphate (ATP)+KSCN. On the other hand, cells of nonhemopoietic origin (e.g., osteoblasts, chondrocytes) were found to be insensitive to ATP4- in this respect. These cells survived the treatment without apparent damage to their alkaline phosphatase activities, osteogenic potentials, and osteoclast induction capacities. The elimination of the endogenous cells of hemopoietic origin from bone tissue or cell populations derived therefrom offers the possibility to study the properties and functions of osteogenic or chondrogenic cells without interference by the presence of cells of hemopoietic origin. It also allows the study of interactions between osteogenic cells and selected cell populations of hemopoietic origin in coculture experiments.

Adenosine Triphosphate↗
PubMed1992

Calbindin immunoreactivity in a subset of cat thalamic reticular neurons.

Recent studies have shown that the thalamic reticular nucleus of cats is made up of several cytoarchitectonically distinct subdivisions and that the nucleus contains accurate topographical maps of the cortical sheet and of the dorsal thalamus. The present study describes immunocytochemically demonstrable heterogeneity in the reticular nucleus of cats, with an antibody to calbindin D28k. The striking feature of calbindin immunoreactivity in the reticular nucleus of cats is that the immunoreactive neurones are located in the caudal half of the nucleus only. In these regions, labelled cells form a small proportion of the total population of reticular cells only and are not distinct in somal size or shape from neighbouring non-labelled reticular cells. Double labelling shows that the calbindin-immunoreactive cells are also immunoreactive to parvalbumin and GABA. There is a distinct tendency for the calbindin-immunoreactive cells to be more numerous ventrally than dorsally in the caudal half of the nucleus, which receives afferents from the somatosensory and auditory systems.

Animals↗
PubMed1976

Dual-labeled technique for human lipid metabolism studies using deuterated fatty acid isomers.

Two deuterated fatty acids, elaidated2 and oleate-d4, were fed simultaneously to a human subject as a mixture of trielaidin-d6 and triolein-d12. Periodically, blood samples were drawn, and red blood cells were separated from the plasma. Red blood cells and plasma lipids were fractionated and analyzed by combined gas chromatography--multiple ion mass spectroscopy. Dual deuterium-labeling allows rate and extent of fatty acid incorporation to be followed in various plasma and red cell neutral and phospholipid fractions. Maximum amount of deuterated fat varied from 4% in cholescurred in either 6-, 8-, or 12-hr samples; generally, less than 1% labeled fatty acids could be detected in 72-hr samples. Because the method is based on dual-labeling, differences in the relative incorporation of both fatty acid isomers can be compared directly. Differences in rates of incorporation, rates of removal, and extent of incorporation of labeled fatty acids into blood plasma can also be determined reliably. Our experimental labeling of fats with deuterium permits for the first time the metabolism of two fatty acid isomers to be compared simultaneously in human subjects. This new method should be applicable to a variety of other lipid metabolic studies.

Adult↗