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At least 973 records · Page 54Linked to original sources
PubMed1983

Aminergic neurons in the anterior nervous system of the rat acanthocephalan Moniliformis dubius.

The cerebral ganglion and nerve tracts of Moniliformis dubius show intense, specific, green fluorescence that is also associated with the lateral and apical sensory bulbs. Radioenzymatic assays showed that high levels of dopamine were present but only small amounts of the catecholamines norepinephrine and epinephrine were identified. Incubations of the proboscis sac in dilute solutions of dopamine increased fluorescence while incubations in reserpine resulted in loss of fluorescence. Nonfluorogenic amine octopamine was also detected radioenzymatically. Neutral red vitally stained a number of cells in the cerebral ganglion and the nerve tracts extending from the ganglion. Electron microscopy showed that many neurons contained electron-dense vesicles. The close association of the fluorescing, amine-containing nerve tracts with the sensory bulbs suggests that they may play a functional role in sensory reception and transmission in M. dubius. This is the first report on the presence of biogenic amines in the Acanthocephala.

Acanthocephala↗
PubMed1984

Dorsal unpaired median neurons, and ventral bilaterally paired neurons, project to a visceral muscle in an insect.

Cobalt backfilling, Lucifer yellow injection and neurophysiological recordings have been used to identify the neurons, in particular dorsal unpaired median neurons, which contribute axons to the oviducal muscles of the locust Locusta migratoria. A total of eight neurons within the VIIth abdominal ganglion have axons passing to the oviducts. Three pairs of bilaterally symmetrical neurons have ventrally located cell bodies. One neuron from each pair projects to the left side of the oviducts and the other the right side of the oviducts. These cells lie ipsilateral to the nerve root through which they exit. The neuropilar branches are intraganglionic and lie mainly in the ipsilateral neuropile, however one of the neurons from each side possesses a giant process, reaching 10 micron in diameter, which passes dorsally to the contralateral side of the ganglion. The other two neurons are dorsal unpaired median neurons, and have large cell bodies which lie at the posterior end of the ganglion. Lucifer yellow injection into these two dorsal unpaired median neurons reveals a single neurite passing anteriorly from the cell body which bifurcates into two bilaterally symmetrical processes which exit to the oviducts through both the left and right sternal roots. Similar to other identified dorsal unpaired median neurons, the cell bodies stain with neutral red and can support overshooting action potentials. The possibility that these two cells contain octopamine is discussed.

Animals↗
PubMed1991

Apoptosis in cultured rat hepatocytes: the effects of tumour necrosis factor alpha and interferon gamma.

We investigated the cytotoxic effects of tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) on rat hepatocytes in culture. Under phase contrast microscopy, we found a small number of dying hepatocytes in control cultures, each having been transformed into a cluster of small spheres. Under transmission electron microscopy, these cells showed the characteristics of apoptosis. TNF alpha and a combination of TNF alpha and IFN gamma exerted a cytotoxic effect, whereas IFN gamma showed no significant cytotoxicity when assessed by neutral red assay and by measuring LDH activity in culture medium. Under phase contrast microscopy, the number of apoptotic cells increased with the addition of either TNF alpha or IFN gamma, and markedly with the addition of both. DNA extracted from apoptotic cells cultured with TNF alpha and IFN gamma was fragmented, and a set of bands of the '200 bp ladder', which is characteristic of the DNA of apoptotic cells, was observed in agarose gel electrophoresis. These findings indicate that cultured hepatocytes die from apoptosis. TNF alpha killed cultured rat hepatocytes by increasing apoptosis, and this effect was potentiated by the addition of IFN gamma, which by itself was also weakly cytotoxic.

Animals↗
PubMed2004

Antiviral activity of Undaria pinnatifida against herpes simplex virus.

The major component of an aqueous extract of the seaweed Undaria pinnati fi da has been identified previously as a galactofucan (GFS), a sulfated polysaccharide. The galactofucan was partially purified and the material tested in this study is 75% pure galactofucan sulfate. GFS was evaluated for antiviral activity against 32 clinical strains of herpes simplex virus (HSV): 14 strains of HSV-1 and 18 strains of HSV-2. Twelve strains (four HSV-1 and eight HSV-2) were resistant to acyclovir (ACV-R) and 20 strains (10 HSV-1 and 10 HSV-2) were susceptible to ACV (ACV-S). The median IC(50) of GFS for the 14 strains of HSV-1 was 32 micro g/mL. The median IC(50) of GFS for the 18 strains of HSV-2 was 0.5 micro g/mL. GFS is significantly more active against clinical strains of HSV-2 than HSV-1, p < 0.001. The mode of action of the GFS was shown to be the inhibition of viral binding and entry into the host cell. The cytotoxicity of GFS was >4.0 mg/mL in the neutral red dye uptake assay indicating that GFS is non-toxic in this assay.

Antiviral Agents↗
PubMed2003

Characteristics of okadaic acid--induced cytotoxic effects in CHO K1 cells.

This article reports the results of investigations into the process of cell death induced in the Chinese hamster ovary cell K1 subclone (CHO K1) by okadaic acid (OA), a hydrophobic polyether produced by marine dinoflagellates. The IC50 was about 13 nM OA after 24 h of treatment, as determined using neutral red. With the MTT assay, the IC50 was 25 nM, although in this case 25% of the initial staining was still observed at 100 nM. Hoechst staining showed that mitotic figures accumulated at 12 nM OA after a 24- or 48-h treatment. In experiments limited to a 3-day treatment without changing the medium, CHO K1 cells were engaged in the death process at 50 nM OA after about 20 h and at 10 nM OA after 48 h. In many cells nuclear fragmentation that resulted in the apparent appearance of vesicles correlated with increasing cellular volume. But additional cell fragmentation was not observed with any treatment, and the chromatin material seemed to progressively disappear inside the cells. DNA fragmentation was analyzed by electrophoresis and with the TUNEL technique. With both techniques, the DNA was fragmented by 48 h in both 25 and 50 nM OA. Electrophoresis showed that both adherent and nonadherent cells were affected. Annexin-positive/ propidium iodide (PI)-negative cells were rarely observed after OA treatment. Some were seen under the scanning cytometer after 20 h at 50 nM OA or after 48 h at 10 nM OA, but they were never detected by flow cytometry. Most of the time scanning cytometry showed either unstained cells or PI-positive (annexin-positive or -negative) cells (48 h, 50 nM, or 72 h, 10 nM). Flow cytometry cytograms showed two cell subpopulations: one composed of a majority of smaller cells, the other of larger cells. The larger cells markedly decreased with time and OA treatment (50 and 100 nM). Stained-cell counting showed that all cells that stained were both annexin- and PI positive and that most PI-positive cells were smaller. Ki67 antigen labeling showed the proliferative activity of CHO K1 cultures but also demonstrated the loss of this activity in smaller cells treated with 50 nM OA for 48 h. We concluded that in our culture conditions the main OA target within CHO K1 cultures was dividing cells. Our results suggest that cells with disturbed metaphase-anaphase enter apoptosis, leading to necrotic daughter cells.

Animals↗
PubMed1995

Reactive oxygen species and DNA damage in 2-bromo-(glutathion-S-yl) hydroquinone-mediated cytotoxicity.

Exposure of renal proximal tubular epithelial cells (LLC-PK1) to the nephrotoxicants 2-bromo-6-(glutathion-S-yl)hydroquinone, 2-bromo-3-(glutathion-S-yl)-hydroquinone, and 2-bromo-(diglutathion-S-yl)hydroquinone caused DNA fragmentation and cytotoxicity. Viability measured by lysosomal neutral red accumulation was the most sensitive parameter of cytotoxicity, and preceded toxicity determined by either the mitochondrial MTT assay or by measuring intracellular lactate dehydrogenase activity. DNA fragmentation was detected as early as 15 min after exposure to 2-bromo-6-(glutathion-S-yl)hydroquinone (100 microM), 2-bromo-3-(glutathion-S-yl)hydroquinone (200 microM), and 2-bromo-(diglutathion-S-yl)hydroquinone (400 microM) and prior to other indices of toxicity. The ability of the cells to repair DNA damage was evident by the decrease in the extent of single strand breaks following removal of 2-bromo-3-(glutathion-S-yl)hydroquinone from the incubation medium. Moreover, inhibition of poly(ADP-ribose)polymerase with 3-amino-benzamide (10 mM), following exposure of LLC-PK1 cells to 0.5 mM 2-bromo-6-(glutathion-S-yl)hydroquinone or 2-bromo-(diglutathion-S-yl)hydroquinone, decreased cytotoxicity, indicating that DNA repair processes, activated in response to DNA damage, exacerbate toxicity. Treatment with the endonuclease inhibitor, aurintricarboxylic acid did not decrease cytotoxicity. A decrease in the cytotoxicity caused by 2-bromo-6-(glutathion-S-yl)hydroquinone and 2-bromo-(diglutathion-S-yl)hydroquinone was observed when cells were incubated with catalase or pretreated with deferoxamine (10 mM). The data suggest a mechanism whereby the conjugates generate hydrogen peroxide, and the subsequent iron-catalyzed generation of hydroxyl radicals causes DNA fragmentation and cytotoxicity.

Animals↗
PubMed2000

The nudel protease of Drosophila is required for eggshell biogenesis in addition to embryonic patterning.

The dorsoventral axis of the Drosophila embryo is defined by a ventral signal that arises within the perivitelline space, an extracellular compartment between the embryo plasma membrane and the vitelline membrane layer of the eggshell. Production of the ventral signal requires four members of the serine protease family, including a large modular protein with a protease domain encoded by the nudel gene. Here we provide evidence that the Nudel protease has an integral role in eggshell biogenesis. Mutations in nudel that disrupt Nudel protease function produce eggs having vitelline membranes that are abnormally permeable to the dye neutral red. Permeability varies among mutant nudel alleles but correlates with levels of Nudel protease catalytic activity and function in embryonic dorsoventral patterning. These mutations also block cross-linking of vitelline membrane proteins that normally occurs upon egg activation, just prior to fertilization. In addition, Nudel protease autoactivation temporally coincides with vitelline membrane cross-linking and can be triggered in mature eggs in vitro by conditions that lead to egg activation. We discuss how the Nudel protease might be involved in both eggshell biogenesis and embryonic patterning.

Animals↗
PubMed1998

Light and scanning electron microscopic cytopathology of 3, 5-dichlorophenol in the permanent fish cell line RTG-2.

The permanent fibrocyte-like fish cell line RTG-2 from rainbow trout (Oncorhynchus mykiss) gonads was investigated by means of light and scanning electron microscopy with respect to alterations as a consequence of sublethal exposure to 0, 1, 10, 16, 25, and 50 mg/liter 3,5-dichlorophenol (3,5-DCP) for 24 h. Control RTG-2 cells were spindle-like in shape and almost three times longer than wide. In subconfluent cultures, they displayed widely spread, flat leading lamellae and formed small groups. Along the edges of the cell protrusions, numerous retraction fibrils could be identified. Except for shallow ridges and small folds, the surface of untreated cells was completely smooth. Following exposure to 3,5-DCP, distinct dose-dependent alterations in cell shape, the appearance of surface blebs and invaginations, as well as progressive retraction of cell extensions could be observed from the lowest test concentration. Morphological changes could be correlated to suppression of lactate dehydrogenase and reduced neutral red retention capacity.

Animals↗
PubMed1998

Mode of cell injury and death after hydrogen peroxide exposure in cultured oligodendroglia cells.

Oxidative stress has been implicated as a causal factor in a wide variety of neurodegenerative diseases. To investigate the direct consequences of oxidative damage on myelin-forming cells, we have exposed oligodendrocytes to hydrogen peroxide. Cytotoxicity was assessed in glial cultures by neutral red (NR) and MTT assay, and half-maximal cytotoxicity was reached after a 30-min application with 100-200 microM H2O2 during a 16-24-h recovery period. The cytotoxic effect could be partly abolished by the simultaneous incubation with N-acetyl-l-cysteine, an antioxidant and precursor of glutathione. In purified mature oligodendroglia cultures (7 div), metabolic activity as determined by the MTT assay, was impaired directly after the treatment with H2O2, and only slightly further enhanced during the 24-h recovery period. Morphological inspection revealed that oligodendrocytes in either the presence or the absence of astrocytes were specifically susceptible to free radical damage, the membranous sheets were disrupted, membranous blebs appeared, and fragmented nuclei were seen. Similar changes were induced by treatment with menadione or staurosporine. The data show that brief exposure to H2O2 induced cell death via apoptosis. This death occurred over a period of 24 h and was accompanied by the appearance of fragmented and condensed DAPI-stained nuclei and internucleosomal DNA cleavage. Concomitantly, as investigated by RT-PCR analysis, the transcriptional activity of c-fos and c-jun was stimulated, without altering mRNA expression of the myelin-specific genes MBP, MAG, and PLP. Thus, oxidative stress in oligodendrocytes leads to the onset of programmed cell death, involving the transcriptional activation of the immediate-early genes c-fos and c-jun.

Animals↗
PubMed1995

A collaborative evaluation of seven alternatives to the Draize eye irritation test using pharmaceutical intermediates.

Much of the data which have been generated on in vitro alternatives to the Draize eye irritation test have dealt with compounds within a specific chemical class or product category. However, in the pharmaceutical industry, it is often necessary to evaluate materials which are not related in structure or properties. It was thus decided to evaluate a diverse series of chemicals in seven in vitro methods for estimating ocular irritation. Thirty-seven test materials were chosen to represent a broad range of pH, solubility, and in vivo irritation potential. Assays were chosen to include as many different types of end points as practical. The group of assays was composed of TOPKAT (assessing structure-activity relationships), bovine corneal opacity-permeability (BCO-P; corneal opacity/toxicity), Eytex (protein coagulation), neutral red uptake (cytotoxicity), MTT in living dermal equivalent (cytotoxicity), Microtox (cytotoxicity in bacteria), and CAMVA (inflammation/toxicity). The results of the study indicated that, in general, the cytotoxicity end points did not correlate well with the in vivo data. The BCO-P, CAMVA, and Eytex assays had the best overall concordance (88.9, 75.8, and 75.0%, respectively) with this set of compounds. Estimation of irritation potential based on structure-activity (TOPKAT) was possible for only approximately 50% of the compounds; however, the assay showed 100% sensitivity (i.e., no false negatives), but low specificity (i.e., negatives correctly identified only 54.5% of the time). These data suggest that for screening of chemicals of diverse structure and properties, the more mechanism-based assays, as opposed to general cytotoxicity assays, hold more promise and should be further evaluated.

Animal Testing Alternatives↗
PubMed1995

Toxicity of four alkylating agents on in vitro rat embryo differentiation and development.

The relative developmental toxicity of four direct acting, alkylating agents was determined in primary cultures of differentiating rat embryo midbrain (CNS) and limb bud (LB) cells and compared with that observed in the rat whole embryo postimplantation culture system. The alkylating agents tested include methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). These alkylating agents have been shown to produce developmental toxicity following either in vitro or in vivo exposure. Viability for both CNS and LB was assessed by a neutral red dye assay. Differentiation of CNS cells was assessed by hematoxylin staining of neurons; differentiation of LB cells was assessed by Alcian blue staining of extracellular proteoglycans. Relative potencies of these compounds in the cell culture system were not the same as those observed in the embryo culture system. Whereas rank order of potency in the cell culture system, for viability and differentiation, was MMS > MNU > ENU > EMS, rank order in the embryo culture system, for embryo lethality and malformations, was MNU > ENU > MMS > EMS. Effective concentrations for cell culture viability and differentiation by MNU and ENU in cell culture were about three to nine times higher than comparable values previously reported for embryos, while effective concentrations for MMS and EMS were two to seven times lower than those observed in the embryos. Differences in potency between the two culture systems may be related to differences in formation and repair of DNA adducts, as well as differences in culture conditions.

Abnormalities, Drug-Induced↗
PubMed1998

FTIR and UV-Vis Spectroscopic Studies of Black Soap Film.

FTIR and UV-vis spectroscopies have been employed to investigate the self-assembly features of a black soap film (BSF) prepared from aqueous solution consisting of 10(-2) M cetyltrimethylammonium bromide (CTAB) and 10(-3) M thiazole yellow (TY) in the liquid-crystalline and gel states, respectively. The construction of two surfactant monolayers of the soap film is independent of the thickness of aqueous core, and the thickness is approximately 1.7 nm at equilibrium. In the liquid-crystalline state, the methylene segments of the hydrocarbon chains are averagely oriented at an angle of 70 degrees with respect to the film normal, and the alkyl chains take a long-range interaction to improve the film elasticity. The TY molecules in the film are horizontally aligned at the monolayer interface in the J-aggregate form. The double-negatively charged TY aggregate greatly enhances the film stability. Dye neutral red is further introduced into the CTAB soap film, the film lifetime is related to the type of the added dyes, in the order of cationic < nonionic < anionic. Copyright 1998 Academic Press.

Journal Article↗
PubMed1997

Regional tissue blood flow and pH in the brain during deep hypothermic retrograde brain perfusion.

Deep hypothermic retrograde brain perfusion is used to protect the brain during aortic arch operations. However, all experiments have failed to demonstrate retrograde blood flow in the brain tissue. We developed an experimental model of sagittal sinus and simultaneous superior vena cava perfusion. Brain tissue blood flow was mapped with colored microspheres during deep hypothermic retrograde brain perfusion in 9 dogs. Regional brain pH was mapped photometrically using neutral red as a pH-indicating dye after 90 min of retrograde brain perfusion in 28 dogs and after 60 min of circulatory arrest in 8 dogs. Cerebral surface blood flow was also measured during retrograde brain perfusion. They were analyzed as functions of driving pressure between sagittal sinus and aorta. Total brain blood flow (ml/min/100 g) was 1.4 +/- 1.3, 3.8 +/- 2.6, and 4.6 +/- 2.6 when the driving pressure was 15, 25, and 35 mmHg, respectively (P < 0.05, 15 mmHg vs 25 mmHg). Regional cerebral blood flow (ml/min/100 g) with a driving pressure of 25 mmHg was 12.1 +/- 9.4, 7.0 +/- 5.6, 4.4 +/- 2.8, and 2.2 +/- 1.4 in the frontal cortex, anterior, mid, and posterior cerebrum, respectively. Cerebral cortex pH was 6.86 +/- 0.23, 7.15 +/- 0.18, and 6.46 +/- 0.13 after 90 min of retrograde brain perfusion with driving pressure of less than 20 mmHg, after that of above 20 mmHg, and after 60 min of circulatory arrest, respectively. Brain tissue pH, blood flows measured with microspheres, and laser flowmetry were highest when driving pressure was between 25 and 35 mmHg. We conclude that retrograde brain perfusion may provide maximum brain protection with driving pressure of 25 to 35 mmHg.

Animals↗
PubMed1999

Apoptosis and morphology in mouse embryos by confocal laser scanning microscopy.

Confocal laser scanning microscopy combined with a vital stain was used to study apoptosis in organogenesis-stage mouse embryos. Apoptosis has previously been visualized in whole embryos using the vital dyes acridine orange, Nile blue sulfate, and neutral red. In the present study, mouse embryos were harvested on Gestation Day 9 and stained with the vital lysosomal dye LysoTracker Red. Following incubation in the stain, embryos were fixed overnight in 4% paraformaldehyde, dehydrated in a graded methanol series, and cleared in benzyl alcohol/benzyl benzoate. The resulting embryo is almost transparent and retains specific LysoTracker Red staining. To achieve optical sectioning through embryos, it was necessary to use low-power objectives. With this procedure, the entire embryo can be optically sectioned and reconstructed in three dimensions to reveal areas of dye staining. Our results demonstrate specific regions undergoing programmed cell death in normal development and increased LysoTracker staining in embryos exposed to hydroxyurea. This procedure allows for the optical imaging of whole Day 9 ( approximately 22 somites) embryos that were greater than 700 microm thick in the z axis and can be applied to studies involving neural tube formation or other aspects of organogenesis.

Aldehydes↗
PubMed1993

ATP depletion is associated with cytotoxicity of a novel lipid regulator in guinea pig adrenocortical cells.

A novel lipid regulator (PD132301-2) produces degeneration and necrosis of adrenal fasciculata in guinea pigs. Primary adrenocortical cell cultures from male Hartley guinea pigs were utilized to investigate potential mechanisms of this toxicity. Concentration-dependent loss of viability, measured by neutral red (NR) accumulation or MTT reduction, was observed within 6 hr at concentrations of 0.01 to 10 microM PD132301-2. At 10 microM, NR and MTT indices were 50% of those of control after 6 hr exposure. Maximal decreases in NR and MTT indices to 20% of control values occurred by 24 hr at > or = 1 microM PD132301-2. Adenine nucleotide analysis after PD132301-2 challenge indicated that ATP depletion preceded loss of viability. At 10 microM PD132301-2, ATP levels were 80% of those of control after 30 min and 25% of those of control after 6 hr. Supplementation of glucose-free buffer with 20 mM fructose protected adrenocortical cells from PD132301-2-induced toxicity. Fructose protection was blocked by inhibiting glycolysis with 1 mM sodium fluoride. Pretreatment of cultures with 100 microM metyrapone, an inhibitor of cytochrome P-450, did not block cytotoxicity induced by 10 microM PD132301-2, but did block cytotoxicity of 100 microM o,p'-DDD. In adrenocortical mitochondrial preparations, inhibition of respiration by PD132301-2 was site II-specific. Both state 3 and state 4 respiration were inhibited 50-75% at 1-30 microM PD132301-2. Thus, ATP depletion resulting from direct inhibition of mitochondrial respiration is a critical early event in adrenocortical cytotoxicity of PD132301-2.

Adenosine Diphosphate↗
PubMed1993

Cytotoxic and genotoxic activity of 1,3-dichloropropene in cultured mammalian cells.

1,3-Dichloropropene (DCP), a widely used soil fumigant previously found to be carcinogenic in both mice and rats, was evaluated for its cytotoxic and genotoxic effects in cultured rodent and human cells. A reduction of cell viability that was dependent on the dose and the length of treatment was observed with the trypan blue and the neutral red assay in both V79 cells and rat hepatocytes exposed to DCP concentrations ranging from 0.18 to 5.6 mM. In the absence of a metabolic activation system, a dose-dependent frequency of DNA single-strand breaks, that were only partially repaired within 24 hr, was revealed by the alkaline elution technique in V79 cells exposed to subtoxic DCP concentrations. The genotoxicity of DCP was confirmed by the results obtained in metabolically competent primary cultures of both rat and human hepatocytes which displayed similar dose-related amounts of DNA fragmentation and DNA repair synthesis, and showed, in comparison to metabolically deficient V79 cells, a somewhat greater sensitivity to the cytotoxic and DNA-damaging effects of DCP. The increase in the frequency of DNA breaks observed in rat hepatocytes after GSH depletion confirms the role of this tripeptide in DCP detoxification; its reduction in hepatocytes simultaneously exposed to metyrapone is consistent with a cytochrome P450-dependent biotransformation of DCP to more toxic metabolites.

Allyl Compounds↗
PubMed1993

Differential cytotoxic sensitivity in mouse and human cell lines exposed to organophosphate insecticides.

Neuroblastoma cell lines were used to examine the differential interspecies response (i.e., species selectivity) to organophosphates (OPs). Baseline activities of the major target esterases, i.e., cholinesterase, carboxylesterase, and neurotoxic esterase, were assayed in mouse and several human neural candidate cell lines. These activities were found to be variable within individual cell lines and among the various tested cell lines. Cytotoxicity data using the neutral red fluorometric assay were collected on both human (SH-SY5Y) and mouse (NB41A3) neuroblastoma clones exposed to a variety of OP insecticides. IC50 data indicated that the tested mouse cell line was consistently more sensitive than the human cell line to equimolar doses of various OP compounds (e.g., mipafox, parathion, paraoxon, DFP, leptophos oxon, fenthion, and fenitrothion). This difference in cytotoxic sensitivity was most pronounced in response to compounds requiring metabolic bioactivation (i.e., protoxicants). Cytotoxicity data also demonstrated that the NB41A3 mouse neuroblastoma cell line was more metabolically competent than the SH-SY5Y human cell line in converting the protoxicant parathion to its neurotoxic metabolite, paraoxon. B-lymphoblastoids, genetically engineered with human P450 cDNAs, demonstrated higher cytotoxic sensitivity to parathion than unengineered cells, indicating that cytochrome P450-associated monooxidase activity could also influence cytotoxic sensitivity to parathion in culture. These data suggest that interspecies-selectivity in response to OP-related cytotoxicity is influenced by intercellular differences in metabolism and baseline esterase activities.

Animals↗
PubMed1994

Effects of ethylnitrosourea on expression of proto-oncogene pp60c-src and high-molecular-weight neurofilament protein in rodent embryo central nervous system cells in vitro.

Effects of exposure to ethylnitrosourea (ENU) on expression of proteins that play a role in neuronal differentiation were examined in central nervous system (CNS) micromass embryo cell cultures. ENU is a known developmental toxicant which affects neuronal development. The proteins selected were the protein product of the src proto-oncogene (pp60c-src) and high-molecular-weight neurofilament protein (NF). pp60c-src has marked increases in amount and kinase activity in neurons at the time of differentiation and NF is found in differentiated neurons. CNS micromass cultures are primary cells from Day 12 rat embryo midbrains which are plated at high density and differentiate into neurons during 5 days in culture. Proteins were quantitated by polyacrylamide gel electrophoresis separation of equal amounts of total cell protein followed by transfer to membranes, immunoblotting, and densitometric scanning of blots. Dose-dependent decreases in cell growth and differentiation were confirmed using endpoints of cell number, protein content of cultures, neutral red uptake, hematoxylin staining of differentiated cells, and levels of binding of the neurotransmitter [3H]gamma-aminobutyric acid. Concentrations which inhibited response by 50% compared to controls ranged from 232 to 455 microM ENU. Dose-related decreases in amounts of pp60c-src and NF proteins relative to total protein were seen in CNS cultures treated with ENU. Results confirm the usefulness of the micromass cultures in following chemical effects on neuronal differentiation. The effects of ENU on specific proteins associated with neuronal differentiation were shown.

Analysis of Variance↗