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At least 991 records · Page 55Linked to original sources
PubMed1995

Modulation of nitrosourea toxicity in rodent embryonic cells by O6-benzylguanine, a depletor of O6-methylguanine-DNA methyltransferase.

Micromass culture systems of mesencephalon (CNS) and limb bud (LB) regions were used to evaluate the impact of the O6-methylguanine-DNA methyltransferase (MGMT) inhibitor, O6-benzylguanine (BG), on ethylnitrosourea (ENU) and methylnitrosourea (MNU)-induced in vitro toxicity. These primary rat embryo cells were preincubated with BG (2.5 microM for CNS; 10 microM for LB) for 24 hr and then ENU (0-1708 microM) or MNU (0-1600 microM) was added. Five days after culture initiation, cell survival was assessed using neutral red uptake and cell differentiation was assessed using selective staining (Alcian blue in LB cultures and hematoxylin in CNS) and image analysis. In both LB and CNS cultures, preexposure to BG resulted in a significant increase in inhibition of differentiation following MNU exposure but no increase in cytotoxicity. There were no statistically significant changes in the toxicity induced by ENU in these differentiating cells consequent to BG preexposure. Our observations lend support to the idea of a specific role for DNA adducts in defining MNU's toxic effects on cell viability and differentiation.

Animals↗
PubMed1996

Effect of antisense oligonucleotides on cytokine release from human keratinocytes in an in vitro model of skin.

ISIS 1082, a phosphorothioate oligonucleotide 21 nucleotides in length targeted to the translation initiation codon of herpes simplex virus (HSV) type 1 and 2 virion capsid protein, has been shown to inhibit HSV-1 replication in vitro. The effects of ISIS 1082, its phosphodiester congener, ISIS 1049, and analogs consisting of 2' methoxy and 2' propoxy phosphodiesters and phosphorothioates on IL-1 alpha release and viability were evaluated in a three-dimensional in vitro skin model consisting of neonatal keratinocytes and fibroblasts. This in vitro system displays many of the functional and metabolic properties of a differentiated epidermis and can be induced to specifically release IL-1 alpha in response to a mixture of lipopolysaccharide and phorbol myristate acetate. Incubation of the skin model with 250 to 1000 microM concentrations of ISIS 1082 and its 2' methoxy and propoxy phosphorothioate analogs resulted in a concentration-dependent increase of cytokine release with minimal effects on cellular viability, as measured by the Neutral Red assay. This response was confirmed in primary keratinocytes, which were also shown to secrete IL-1 alpha into media supernatants after incubation with phosphorothioate oligomers. These data suggest that the IL-1 alpha released from keratinocytes in response to ISIS 1082 may contribute to the inflammatory and immune cell response seen in vivo.

Antiviral Agents↗
PubMed2002

Impairment of the cell-to-matrix adhesion and cytotoxicity induced by Bothrops moojeni snake venom in cultured renal tubular epithelia.

Bothrops moojeni snake venom induces acute renal failure (ARF) as a consequence of morphological and functional alterations in glomerular and tubular cells. It is still unclear whether the ARF results from a direct cytotoxic effect on renal epithelia or from a renal ischemia due to systemic hemodynamic disturbances. This work investigated the in vitro effect of B. moojeni crude venom, using cultured Madin-Darby canine kidney (MDCK) monolayers as a model. The crude venom induced a significant time- and dose-dependent decrease in transepithelial electrical resistance across MDCK monolayers. In addition, the exposure to the venom resulted in cell detachment from the substratum, as revealed by transmission electron microscopy. Immunocytochemical analysis showed no change in the distribution of some junctional proteins, such as occludin, ZO-1, and E-cadherin. Nevertheless, the staining with labeled phalloidin revealed a disarray of the cytoskeleton, specifically of the stress fibers and of the focal adhesion-associated F-actin at the cell-to-matrix contact region. The treatment with B. moojeni venom also increased the cell release of lactate dehydrogenase and decreased cellular uptake of the vital neutral red. In conclusion, B. moojeni crude venom appears to have a direct cytotoxic effect on a renal tubule-derived cell line, also inducing impairment of the cell-matrix interaction.

Animals↗
PubMed2003

Induction of apoptosis by vitamin D metabolites and analogs in a glioma cell line.

Gliomas are the most common malignant tumors in brain. Recent studies demonstrate the capacity of 1alpha,25(OH)2D3 to specifically induce cell death (apoptosis) in model glioma cell lines and in primary cultures from tumor tissue, but not in primary astrocytes. In spite of this promising activity, a broad therapeutic application of vitamin D metabolites and analogs is still restricted because of their poor bioavailability and their hypercalcemic actions. Compared to 1alpha,25(OH)2D3, its natural 3alpha-epimer exhibits far higher metabolic stability and a reduced calcemic effect. Focusing on a possible therapeutic advantage of the 3alpha-conformation, we have examined the apoptotic potential of a representative set of vitamin D analogs, each of them in the 3alpha- and 3beta-conformation, and of natural vitamin D metabolites in the rat C6 glioma cell line. Exposure of these cells to the synthetic analogs resulted in all cases in a pronounced reduction of cell density (tested by incorporation of neutral red) and induction of apoptosis, monitored by staining nuclei with Hoechst 33258 dye and by following DNA fragmentation by capillary electrophoresis. The 3alpha-epimers showed equivalent or even higher activity on C6 cells than their respective 3beta forms. For their potent effects on growth and apoptosis of tumor cells and their high metabolic stability combined with a low calcemic potential, we speculate that these 3a-epimers could provide advantages for a prospective treatment of glioma.

Animals↗
PubMed1987

In vitro cytotoxicity of heavy metals, acrylamide, and organotin salts to neural cells and fibroblasts.

The cytotoxicity of neurotoxic agents was determined for a series of brain-derived cell types and compared with their toxic effects on BALB/c 3T3 fibroblasts, using the neutral red assay. Ranking of toxicants according to their potencies was the same for all cells tested and was in the order of methylmercury greater than cadmium greater than mercury greater than zinc greater than acrylamide. For a series of di- and triorganotins the ranking order was dibutyl greater than diphenyl greater than dibenzyl greater than dipropyl greater than diethyl greater than dimethyltin and triphenyl greater than tribenzyl greater than trimethyltin, respectively. The test was sensitive enough to detect structure activity relationships between the degree of toxicity and the hydrophobic characteristics of the agents tested.

Acrylamides↗
PubMed1992

A histofluorescent study of sympathetic innervation of human palatine tonsils.

The glyoxylic catecholaminergic histofluorescence method was employed on human palatine tonsil specimens in order to study the sympathetic innervation present. One percent neutral red was used as a counterstain. Abundant sympathetic fibers were demonstrable around the blood vessels of the medulla and capsule. However, few sympathetic fibers were found around the vessels of the subepithelial connective tissue and interfollicular septa. In the areas of the follicle and extrafollicle where B and T lymphocytes were located, sympathetic fibers were not found. These findings indicate that if sympathetic innervation can affect T and B cells, it will do so indirectly. Results also show that there is a higher norepinephrine content in focally infected tonsils that is not due to hyperactivity of the sympathetic nerve, but may be due to other mechanisms. Finally surgical dissection at the capsule during tonsillectomy will reduce bleeding, perhaps because vessels there have an abundant sympathetic innervation that leads to good vessel contraction.

Adrenergic Fibers↗
PubMed1994

Sympathetic innervation of the eustachian tube in rats.

The glyoxylic catecholaminergic histofluorescence method was employed on the mucosa of the rat's eustachian tube (ET) in order to study the sympathetic innervation present. One percent neutral red was used as counterstain. Many noradrenergic fibers were demonstrable around blood vessels, glands and submucosa of the ET, but not in the epithelium. In a group of rats following neurectomy, the superior cervical ganglia (SCG) were removed unilaterally or bilaterally. Changes in sympathetic innervation of the ET were examined 14 days after SCG ganglionectomy. In those animals after unilateral SCG ganglionectomy, no noradrenergic histofluorescence was found in the ipsilateral ET, although some scant fluorescence could be detected in the tube's nasopharyngeal (NP) orifice. However, no noradrenergic histofluorescence could be observed in animals bilateral SCG ganglionectomies. Our results indicate that sympathetic innervation of the ET in the rat originates in the SCG, with some cross-innervation of sympathetic fibers occurring in the tube's NP orifice.

Animals↗
PubMed1987

Human non-malignant and malignant brain tumor derived cell cultures: proliferation and sensitivity to natural human fibroblast (beta) interferon.

Twenty-one human brain tumor biopsies were processed by mechanical and enzymatic methods to produce mixed cell suspensions. Cultures were prepared in small plastic flasks, and primary outgrowth occurred in 16/21 cultures. The period required for primary outgrowth ranged from 3 days to 14 days. We established serial propagation with 15/16 of the primary cultures. Sensitivity to HuIFN-beta was determined between passages 3 to 12, using a microassay based on cell viability (uptake of a supravital stain, neutral red). Extracted dye was quantified in acidic-methanol using the MR580 Microelisa Autoreader (Dynatech). We observed a broad range of responsiveness to the drug among the 12 cell-strains tested. Thus, 4 cell strains were relatively sensitive; 4 were resistant to 10(4) IRU/ml of purified HuIFN-beta. Four cell strains exhibited a level of responsiveness that was intermediate to that of these two groups. During propagation of these biopsies, cytopathology suggestive of paramyxovirus-infection appeared in 4 of the cell-strains. This characteristic was not uniformly associated with high sensitivity to human beta interferon which is a very potent, naturally occurring antiviral substance. Our results support the concept that information concerning sensitivity to HuIFN-beta and other cytostatic agents may be rapidly obtained using microcultures of brain tumor cultures in conjunction with supravital stain uptake studies. Additionally, these results suggest that further clinical studies with beta interferon should be undertaken to define the parameters which determine successful in vivo application.

Astrocytoma↗
PubMed1996

Inhibitory effects of heavy metals on cytochrome P4501A induction in permanent fish hepatoma cells.

The interactions in vitro of heavy metals Cd(II), Co(II), Cu(II), Ni(II), Pb(II), and Zn(II) with cytochrome P4501A (CYP1A) induction response and enzyme activity were studied in fish hepatoma cells PLHC-1. Cells were simultaneously exposed to heavy metals and to 3-methylcholanthrene (3-MC), an inducer of CYP1A. Heavy metals were added to the cells in different concentrations. Cytotoxicity were measured in the neutral red (NR) assay, relative CYP1A protein contents in an enzyme-linked immunosorbent assay (ELISA), and CYP1A activities in the ethoxyresorufin-O-deethylase (EROD) assay. All metals had a more pronounced effect on EROD activity than on CYP1A protein content and cytotoxicity. For the most active metal Cd(II), a 50% inhibition of EROD activity was observed at significantly lower concentrations (2.2 x 10(-5) M) than a 50% reduction of CYP1A protein (5.3 x 10(-5) M), and a 50% cytotoxicity (1.4 x 10(-4) M). The inhibitory potency of the metals had the following order: Cd(II) > Ni(II) > Cu(II) > Co(II) = Zn(II) > Pb(II). In a second set of experiments, lysates of 3-MC-induced cells were exposed to heavy metals. Cd(II) and Cu(II) caused a 50% inhibition of EROD activity at significantly lower concentrations than in the experiments with living cells, at 8.2 x 10(-6) M and 1.3 x 10(-5) M, respectively, whereas the effect by Co(II) occurred at a significantly higher concentration (8.2 x 10(-4) M). The results indicate that Cd(II) and Cu(II) in particular may affect the CYP1A system of the liver of fish at low concentrations through direct inhibition of the CYP1A enzyme activity. CYP1A induction response in fish liver is increasingly being used in biomonitoring programs. In the environment, interactions of CYP1A-inducing and CYP1A-inhibiting components (such as heavy metals) can be expected and must be taken into consideration.

Animals↗
PubMed1989

Fusion of epithelial sheets as seen in formation of the chick amnion.

Fusion of the amniotic folds over the chick embryo provides an example of epithelial fusion in which one group of ectodermal cells appears to contribute to traction and a second group appears to respond by moving to the midline, becoming internalized and degenerating. Neutral red staining of living embryos and transmission electron microscopy indicate that a patch of dying cells is present in the fusion region. Scanning electron microscopy shows that ectoderm cells at the rim of the amniotic folds are cylindrical in shape with long axes oriented parallel to the rim. These cells have a smooth surface, but show increasing amounts of blebbing toward the midline. Characteristically, a globular region of rounded cells showing extreme blebbing is present at the midline. Just anterior to the globular region, flattened cells are present on the surface with many taut cellular processes. Orientation of ectodermal cells around the region of fusion suggests that traction is exerted from these flattened cells. When treated with cytochalasin D, posterior progression of the zone of fusion is blocked. Also, the flattened cells anterior to the globular region round up, revealing many elongated, anteriorly-directed cytoplasmic processes presumably leading to sites of original attachment.

Amnion↗
PubMed1993

Reduced light sensitivity of the circadian clock in a hypopigmented mouse mutant.

Pink-eyed dilution (p/p) is a recessive mutation in mice which results in reduced pigmentation of the retinal pigment epithelium, as well as alterations in visual pathways and function. We investigated whether this mutation also affects light information reaching the circadian clock. Entrainment to a 12 h light 12 h dark cycle and the free-running period in constant darkness were not affected by this mutation. Phase shifts in response to 1 h light pulses consisting of bright white light at either circadian time 16 or 24 also did not differ between mutant and wild-type C57BL/6J mice. However, when 5 min, 502 nm light pulses of 1.2 x 10(-1) microW/cm2 or 4 x 10(-2) microW/cm2 were given at circadian time 16, the mutant mice responded with significantly smaller phase shifts than the wild-type mice. When animals were transferred to constant light, the free-running period of wild-type mice was longer than that of mutant mice, a finding which is consistent with a sensitivity difference between mutant and wild-type mice. Horseradish peroxidase tracing of retinal innervation of the hypothalamic suprachiasmatic nuclei (SCN)--the location of a circadian pacemaker--revealed a reduced innervation of the SCN in mutant mice compared with wild-type mice. The total volume of the SCN, as determined by neutral red stain, was also reduced in mutant mice, although not to as great an extent as the retinal innervation. Taken together, these results indicate that while basic characteristics of circadian clock function are not altered by the pink-eyed dilution mutation, the sensitivity of the clock to light is reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1979

Studies on the induction of petite mutants in yeast by analogues of berenil. Characterization of three mutants resistant to the compound Hoe 15,030.

Compound Hoe 15,030 is an analogue of berenil which is as effective as berenil in inducing petite mutants in Saccharomyces cerevisiae. Hoe 15,030 has greater stability than berenil in aqueous solution, and is less toxic to yeast at high drug concentrations. Mutants of S. cerevisiae strain J69-1B have been isolated which are resistant to the petite inducing effects of Hoe 15,030. Three mutant strains (HR7, HR8 and HR10) were characterized and each was shown to carry a recessive nuclear mutation determining resistance to Hoe 15,030. The degree of resistance to Hoe 15,030 is different for each mutant, and each was found to be co-ordinately cross-resistant both to berenil and to another analogue of berenil, Hoe 13,548. However, the three mutants show no cross-resistance to other unrelated petite inducing drugs, including ethidium bromide, euflavine and 1-methyl phenyl neutral red. Further studies on the mutants revealed that each strain exhibits characteristic new properties indicative of changes in mitochondrial membrane functions concerned with the replication (and probably also repair) of mitochondrial DNA. Thus, mutant HR7 is hypersensitive to petite induction by the detergent sodium dodecyl sulphate under conditions where the parent J69-1B is unaffected by this agent. Mutant HR8 is even more sensitive to sodium dodecyl sulphate than is HR7, and additionally shows a markedly elevated spontaneous petite frequency. Isolated mitochondria from strains HR8 and HR10 (but not HR7) show resistance to the inhibitory effects of Hoe 15,030 on the replication of mitochondrial DNA in vitro.

DNA Replication↗
PubMed1990

High endocytotic and lysosomal activities in segments of rat myotubes differentiated in vitro.

Endocytosis and the lysosome system have been studied in rat myotubes differentiated in vitro. Horseradish peroxidase was used as marker for endocytosis and was found to accumulate unevenly in the myotubes. Small segments of myotubes display very high endocytotic activity. Similar segments contained numerous lysosomes, as seen by the accumulation of neutral red or histochemical staining for acid phosphatase. The segments also contained accumulations of acetylcholine receptors as determined by binding of tetramethyl rhodamine-labelled alpha-bungarotoxin. Unstained segments in living cultures could be recognized by phase-contrast microscopy since they often appeared somewhat dilated and were not as well spread on the culture surface as the main parts of the myotubes. Ultrastructurally, the segments contained an intensely proliferating tubular system in communication with the extracellular space, which therefore probably represents the developing transverse tubular system. The segments also contained endocytosed marker within large phagosomes. Contractile filaments occurred in the segments but were frequently less well-organized than in other parts of the myotubes. The described characteristics of the segments in rat myotubes differentiated in vitro bear resemblance to some of the characteristics of the denervated endplate region of adult muscle.

Animals↗
PubMed1988

Response of cultured rat Kupffer cells to lipopolysaccharide.

The effects of Salmonella abortus equi lipopolysaccharides (LPS) on pure cultures of rat Kupffer cells (Kc) were studied. In vitro, LPS is ingested by Kc and located in vacuoles and secondary lysosomes. Culture of Kc in the presence of 1-50 micrograms LPS/ml during 24 h did not affect the viability of the Kc as measured by trypan blue exclusion, neutral red uptake, lactate dehydrogenase leakage and cell survival and spreading. LPS treatment did not influence the ultrastructure of Kc. The exposure of Kc to LPS in vitro did not change the phagocytic activity. Several biochemical processes were stimulated: glucose consumption, MTT-tetrazolium salt reduction, total protein synthesis and secretion of proteins. LPS activated Kc to tumoricidal activity against L929 mouse fibrosarcoma cells. In the light of the above observations, it is concluded that purified LPS is not cytotoxic for pure Kc in culture. On the contrary, LPS stimulates several biochemical and functional processes.

Animals↗
PubMed1995

In vitro evaluation of donor liver preservation fluids on human hepatocyte function.

Successful liver transplantation depends on adequate preservation of cellular function. We therefore tested the effects of two currently used liver preservation fluids, Euro-Collins (EC) solution and University of Wisconsin (UW) solution, on the viability and some functional activities of hepatocytes isolated from human livers. Cells in primary culture were maintained under hypoxic (95% N2/5% CO2) and hypothermic (4 degrees C) conditions for 24 h, either in EC or UW solution. This treatment did not result in significant hepatocyte damage, as judged by phase contrast microscopy, intracellular LDH release, and the MTT mitochondrial test. However, neutral red uptake indicated that lysosomal functions were slightly affected (35% decrease) when compared to control conditions. At the end of the hypoxia/hypothermia period, hepatocyte monolayers were incubated at 37 degrees C under normoxic conditions for 24 h, in order to simulate the reperfusion of a transplanted liver. Three drugs--midazolam, diazepam, zidovudine--were used as diagnostic substrates to check the metabolic abilities of human hepatocytes replaced in normal conditions. Both phase I (hydroxylation, demethylation) and phase II (glucuronidation) metabolic reactions were affected by the hypoxia/hypothermia shock. Indeed, a 30%-50% decrease in these activities was observed as compared to values obtained in control hepatocytes. No difference could, however, be found at the cellular level regarding the solution used for cold storage. These results suggest that the superiority of UW over EC solution, already reported in clinical practice after transplantation of preserved human livers, was not due to a better preservation of the hepatocytes.

Antimetabolites↗
PubMed1991

Cross contamination associated with the use of multiwell culture plates for cytotoxicity assessment of volatile chemicals.

In vitro toxicity testing can involve technical problems due to the evaporation of volatile test chemicals. The cytotoxicity of two volatile chemicals (butanol and ethanol) has been assessed with neutral red assay in conventional microtiter plates. The non volatile DMSO chemical is used as a negative control. Under these conditions, an important cross contamination between test concentration groups has been observed. This affects cytotoxicity estimation which is overestimated. This cross contamination is prevented when special plates containing removable bars are used.

Butanols↗
PubMed1976

Histochemical characteristics of the egg envelopes of Acanthosentis sp. (Acanthocephala).

The histochemical and phase contrast observations on the embryonic envelopes enclosing the acanthor of Acanthosentis sp. reveal the presence of three layers. All the three layers were non-refractile during early stages of their formation, and become refringent subsequently. The outer layer loses its refractile property together with affinity for acid and basic dyes when the eggs are fully developed. This layer is neither tanned nor chitinous. The second layer consists of glyco-lipo-proteinous matrix. The polysaccharide moiety is identified to be strongly sulfated acid mucopolysaccharide. The lipid component has been characterized to be KOH resistant and esters of unsaturated fatty acid. The protein is rich in aromatic amino acids. The third and the innermost layer contains chitin and acid mucopoly-saccharides. The protein is rich in sulfur-containing amino acids. Sulphydryl groups were present during early stages of its formation, but forms disulphide cross-links subsequently. Stages in the development of acanthors were characterized with reference to the formation of envelopes. The permeability experiments carried out on fresh eggs using 0.1% aqueous solutions of bromophenol blue, methylene blue, toluidine blue, basic fuchsin and neutral red indicate that the inward diffusion of these substances is prevented subsequent to formation of disulphide groups in the innermost layer. The results further indicate that the embryo derives nutritive requirements from the fluid of pseudocoel of the female worm through th envelopes covering it.

Acanthocephala↗
PubMed1996

An in vitro nucleoside analog screening method for cancer gene therapy.

Suicide genes that sensitize cells to drugs that are normally nontoxic at therapeutic levels represent an important approach in human gene therapy research. We have developed an in vitro screening assay to assess the modulation of nucleoside analogs after transfection of a vector expressing the herpes simplex virus thymidine kinase gene (HSV-TK). The thymidine kinase gene enhances nucleoside phosphorylation to nucleotides that kill cells by blocking DNA elongation. Cells lines used are 3T3-NIH fibroblasts (parental cells) and 3T3-TKc3 (HSV-TK gene-transfected 3T3-NIH). Two types of analysis are performed: a cytotoxicity assay, the neutral red uptake assay to assess the IC50 on the two cell lines, and an HPLC analysis coupled to a radiochemical flow detector to evaluate metabolic profiles after incubation of cells with tritiated analogs. Results show that cells expressing the HSV-TK gene are more sensitive than the parent cells to the effect of acyclovir or ganciclovir, the reference purine analog drugs, and also to the effect of pyrimidine analogs, bromodeoxyuridine, bromovinyldeoxyuridine, and ethyldeoxyuridine. Promising nucleoside analogs for gene therapy that can be achieved by HSV-TK could be evaluated using this model.

3T3 Cells↗