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Hypoxia produces cell death in the rat hippocampus in the presence of an A 1 adenosine receptor antagonist: An anatomical and behavioral study

Endogenous adenosine depresses synaptic transmission in rat hippocampal slices during periods of hypoxia, a potentially neuroprotective cellular response that is attenuated by the adenosine antagonist 8-cyclopentyltheophylline. In this study, rats were exposed to moderate hypoxic conditions (5% O 2−95% N 2, 40 min × three days) in the presence or absence of 8-cyclopentyltheophylline, and the effects of reducing adenosinergic inhibition during hypoxia were assessed histologically and behaviorally. Half the rats exposed to low levels of oxygen in the presence of 8-cyclopentyltheophylline (10 mg/kg) suffered unilateral or bilateral hippocampal damage largely limited to the CA1 subfield. Animals which had suffered hippocampal damage were also impaired in their performance of a working memory version of the Morris Water Maze, but not a passive avoidance task (step-through). Hypoxia alone did not result in neuronal damage or behavioral impairment. These results provide further evidence that endogenous adenosine provides an important level of neuronal protection during even prolonged periods of hypoxia.

NIH workshop on experimental spinal cord injury: modeling and criteria.

Journal of NeurotraumaVol. 9, No. 2 NIH Workshop on Experimental Spinal Cord Injury: Modeling and CriteriaDOUGLAS K. ANDERSON, CHUNG Y. HSU, MARY ELLEN MICHEL, and BRADFORD T. STOKESDOUGLAS K. ANDERSONSearch for more papers by this author, CHUNG Y. HSUSearch for more papers by this author, MARY ELLEN MICHELSearch for more papers by this author, and BRADFORD T. STOKESSearch for more papers by this authorPublished Online:26 Mar 2009https://doi.org/10.1089/neu.1992.9.113AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byAcute Cellular and Functional Changes With a Combinatorial Treatment of Ion Channel Inhibitors Following Spinal Cord Injury25 June 2020 | Frontiers in Molecular Neuroscience, Vol. 13Granzyme-b mediated cell death in the spinal cord-injured rat modelNeuropathology, Vol. 29, No. 3Injury in the spinal cord may produce cell death in the brainBrain Research, Vol. 1020, No. 1-2Alterações vesicais na lesão medular experimental em ratosActa Cirurgica Brasileira, Vol. 18, No. 3Animal and Culture Models of Glaucoma for Studying Neuroprotection18 January 2018 | European Journal of Ophthalmology, Vol. 11, No. 2_supplBehavioral and Histological Outcomes Following Graded Spinal Cord Contusion Injury in the C57Bl/6 MouseExperimental Neurology, Vol. 169, No. 2Modeling axonal injury in vitro: injury and regeneration following acute neuritic traumaJournal of Neuroscience Methods, Vol. 102, No. 1Spinal cord injury in the ratProgress in Neurobiology, Vol. 56, No. 3Chapter 23 Influence of dynorphin A antibodies on the formation of edema and cell changes in spinal cord traumaAn Electromechanical Spinal Injury Technique with Dynamic Sensitivity BRADFORD T. STOKES, DAVID H. NOYES, and DONALD L. BEHRMANN26 March 2009 | Journal of Neurotrauma, Vol. 9, No. 3Protective Effect of Methylprednisolone on Vascular Injury in Rat Spinal Cord Injury J. XU, Z.X. QU, E.L. HOGAN, and P.L. PEROT26 March 2009 | Journal of Neurotrauma, Vol. 9, No. 3 Volume 9Issue 2Jan 1992 To cite this article:DOUGLAS K. ANDERSON, CHUNG Y. HSU, MARY ELLEN MICHEL, and BRADFORD T. STOKES.NIH Workshop on Experimental Spinal Cord Injury: Modeling and Criteria.Journal of Neurotrauma.Jan 1992.113-116.http://doi.org/10.1089/neu.1992.9.113Published in Volume: 9 Issue 2: March 26, 2009PDF download

The Role of Cell Proliferation in Multistage Carcinogenesis

Carcinogenesis is a complex process in which it is believed that normal cellular growth control genes are altered by sequential mutational events, with subsequent clonal growth of the resulting precancerous or cancerous cells (1, 2). The induction of mutations and the preferential clonal growth of the resulting premalignant or malignant cells, thus, become critical events in the stages of initiation, promotion, and progression in chemical carcinogenesis (Fig. 1). One class of chemical carcinogens are the genotoxicants. These compounds or their metabolites are DNA reactive and directly induce mutations or clastogenic changes. The observation that most mutagens are also carcinogenic is the basis for many current predictive assays and risk assessment models. However, there are also different classes of nongenotoxic carcinogens that do not interact with the DNA. The class designated as mitogens directly induces cell proliferation in the target tissue. Another class, the cytotoxicants, produces cell death foll...

Modulation of interleukin-2-driven proliferation of human large granular lymphocytes by carbaryl, an anticholinesterase insecticide

Studies in other laboratories have provided evidence that the interleukin-2 (IL2) signaling pathway in lymphocytes includes essential, serine proteases. Since the anticholinesterase (antiCHE) insecticides are potent serine hydrolase (esterase and protease) inhibitors, we assessed the ability of carbaryl (CA, a widely used antiCHE insecticide) and α-naphthol (NA, a major metabolite of carbaryl) to modulate IL2-driven proliferation of large granular lymphocytes (LGL) purified from human peripheral blood. These cells express nearly all of the natural killer (NK) activity of human peripheral blood. NK cells are normal lymphocytes that respond to IL2 by proliferating and increasing their tumoricidal activity on a per cell basis. Cultures of purified LGL, initiated in the presence of human recombinant IL2, were harvested on culture Day 4, then proliferation was measured as [ 3H]thymidine incorporation. When added only at the time cultures were initiated, CA inhibited incorporation 10–32%, 35–53%, and 54–57% at 0.5, 5.0, and 50 μ m, respectively. In contrast, NA had no effect at 0.5 and 5.0 μ m, but was inhibitory (16–17%) at 50 μ m. Reexposure to CA or NA, during the incorporation assay, had little effect on the observed inhibition profiles. Chemically induced changes in cell number during an 8-day culture period reflected the chemically induced changes in [ 3H]thymidine incorporation. Neither CA nor NA produced cell death. Quantitation of both CA and NA by HPLC indicated a rapid loss of CA (ca. 95% in 24 hr) and a much slower loss of NA from the culture medium. CA inhibited human LGL proliferation at concentrations producing little or no inhibition of serum CHE, an indicator of exposure to antiCHE insecticides.

Possible role of cell cycle-dependent morphology, geometry, and mechanical properties in tumor cell metastasis

Studies that examine the shear- and abrasion-sensitivity of proliferating cells are important in order to understand the behavior of hybridoma cells in bioreactor culture and metastasizing cancer cells in the bloodstream. Little is known about the link between morphology, structure, and mechanical properties of a given cell line, especially with respect to variations throughout the cell cycle. In our experiments with GAP A3 hybridoma cells, distinct cell morphologies were identified and correlated with phases of the cell cycle by video microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (apparent cell viscosity) properties of single cells. As the cell cycle progressed at 37 degrees C, an increase in cell volume from 1400 microns 3 to 5700 microns 3 was accompanied by an increase in apparent cell viscosity from 430 poise to 12,000 poise, consistent with an accumulation of more cytoplasmic material in the "older" cells. Hybridomas are representative of the various leukemias derived from hemopoietic cells, and even though as a whole, they appeared to be rather shear-insensitive, the wide range of property values demonstrates that a given cell line cannot be characterized by a single value for any one property, and that properties must be related to the cell cycle when considering proliferating cells. It is interesting to see if distinct stages in the metastatic sequence of events might correlate with any of these physical features of the cell cycle, irrespective of cell type or cell line. For example, the cytokinetic doublet could represent a fragile structure that may fail and produce cell death under fluid-shear conditions that would not affect the cells at any other stage in the cell cycle. Identifying such cell cycle-dependent features in metastasizing cancer cells could lead to a better understanding of the metastatic process and to possible clinical treatments directed at making cells more shear- and abrasion-sensitive, and therefore, more likely to be killed by the natural hydrodynamic forces of the circulatory system.

Human epithelial ovarian cancer cell steroid secretion and its control by gonadotropins

To elucidate the role of gonadotropins in regulating steroid metabolism in human epithelial ovarian carcinoma (OV Ca), cells were cultured from a number of OV Ca localized to the ovary. These cells uniformly secreted 17-β-estradiol (E 2), and cells from some OV Ca also secreted progesterone (P), as well as CA 125. Secretion rates decreased with time in culture and number of subcultures. In the original and first few subcultures, 1–10 pg/ml/μg DNA/24 hr of E 2 was secreted and P secretion varied from 1 to 8 ng/ml/μg DNA/24 hr under basal conditions. Secretion rates for CA 125 were between 5 and 300 U/ml/day. Approximately 30% of the primary cultures from cystadenocarcinomas responded to hCG and hFSH and 70% of cultures responded to 8-Br-cAMP with 2- to 10-fold increases in secretion of E 2. In one primary culture, hCG produced a dose-related increase in E 2 production between 1 and 5 ng/ml, but the response declined to zero at 25 ng/ml. In the same cells, exposure to hCG and cAMP for 72 hr produced cell death, whereas hFSH had no such effect. Subculturing reduced steroidogenic responses to the hormones but the response to cAMP was maintained to a greater degree. These results suggest that some OV Ca-derived cells are steroidogenic in vitro and that some of these cells respond with increased E 2 secretion to agents which are well-known stimulators of steroidogenesis in normal ovarian cells.

Structure-function relations in radiation damaged DNA.

The most important biological effects of exposure to ionizing radiation can be related to a variety of changes in cell function. Some of these changes can produce cell death, but others lead to less final deleterious effects such as carcinogenesis or altered cell function as a result of energy deposition in the biological system. All the changes in cell function can be linked to DNA damage, with the double-strand break and the radiation-induced mutations causing most of the lethal damage. Increasingly more accurate and direct measurements in radiation dosimetry, as discussed at this Conference, and the understanding provided by the theories and formulations of condensed matter physics, also presented and discussed at great length at this meeting, have offered important insight into the parameters and measurable outcomes of exposure to ionizing radiation. These are enhanced by findings, such as those presented at this Conference by Clemens von Sonntag, that emerge from radiochemistry measurements in vitro of chemical changes produced by radiation exposure. Also as described at this conference by Aloke Chatterjee and Herwig Paretzke, computational simulations based on Monte Carlo algorithms have been developed to explore the parameters of the energy deposition processes and their consequences in models of the biological systems. But a clear, mechanistic link between the physical processes and the biological consequences remains somewhat elusive, suggesting that it will be necessary to know and understand first the sequence of events that lead from energy deposition of radiation in condensed matter to the biophysical and biochemical processes that occur at the level of cellular DNA.

Toxicity of N-(3,5-dichlorophenyl)succinimide and metabolites to rat renal proximal tubules and mitochondria

The acute nephrotoxicity caused by N-(3,5-dichlorophenyl)succinimide (NDPS) has been shown to be due to a metabolite(s) of the parent compound. This study examined the toxicity of NDPS, its known metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA), N-(3,5-dichlorophenyl)malonamic acid (DMA), N-(3,5-dichlorophenyl)succinamic acid (NDPSA), and two postulated metabolites N-(3,5-dichlorophenyl)maleamic acid (NDPSA) and N-(3,5-dichlorophenyl)maleimide (NDPM) to suspensions of renal proximal tubules (RPT) prepared from male Fischer 344 rats. Tubule viability and mitochondrial function were not adversely affected by exposure of RPT to either 1 mM NDPS, NDHS, NDHSA, DMA, NDPSA, or NDPMA for 4 h. However, NDPM caused a concentration-(25–100 μM) and time-dependent (0.25–4 h) loss in basal and nystatin-stimulated oxygen consumption and tubule viability. Investigations using isolated renal cortical mitochondria (RCM) showed that NDPM was a potent inhibitor of mitochondrial function. Isolated RCM respiring on pyruvate/malate and exposed to NDPM exhibited a concentration (25–100 μM) dependent decrease in state 3 and state 4 respiration. Inhibition of mitochondrial state 3 respiration by NDPM was mediated through site 1 of the respiratory chain. NDPM did not inhibit cytochrome c-cytochrome oxidase or the electron transport chain. These results indicated that NDPS, its known metabolites, and NDPMA were not directly toxic to rat RPT. However, the postulated metabolite NDPM, was a potent tubule cytotoxicant that inhibited mitochondrial function in isolated RCM and RPT and may produce cell death through this mechanism.

Maitotoxin-induced liver cell death involving loss of cell ATP following influx of calcium

Maitotoxin, one of the most potent marine toxins known, produced cell death in cultures of rat hepatocytes with a TD50 of 80 p m at 24 hr. The cell death, as indicated by a dose- and time-dependent leakage of lactate dehydrogenase (LDH), was also associated with the leakage of [ 14C]adenine nucleotides from hepatocytes prelabeled with [ 14C]adenine. The toxic effect of maitotoxin was completely abolished by the omission of calcium from the culture medium. The cell death induced by maitotoxin increased with increasing concentrations of calcium in the medium. Treatment of hepatocytes with low concentrations of the toxin (<0.5 ng/ml) resulted in increases in 45Ca influx into the cells. At higher concentrations of maitotoxin (>1 ng/ml), the initial increase in 45Ca influx was followed by the release of the 45Ca from the cells into the medium. Since the 45Ca release paralleled the LDH leakage, the release of calcium was due to cell death. The 45Ca influx, [ 14C]adenine nucleotide leakage, and LDH leakage were effectively inhibited by verapamil, a calcium channel blocker. Maitotoxin also induced a time- and dose-dependent loss of ATP from hepatocytes, which preceded the [ 14C]adenine nucleotide and LDH leakage. Thus, it appears that the cell death resulting from maitotoxin treatment is caused by the elevated intracellular calcium, which in turn inhibits mitochondrial oxidative phosphorylation causing depletion of cell ATP. Loss of cell ATP may be the causative event in the maitotoxin-induced cell death.

Spark decomposition of SF/sub 6/: chemical and biological studies

Because electric arcs, sparks or corona can decompose SF/sub 6/ insulators into byproducts having chemical properties different from SF/sub 6/, environmental concerns arise regarding inadvertent human exposures to electrically decomposed SF/sub 6/. Biological assays using mammalian cell culture systems have revealed that SF/sub 6/, spark-decomposed under specific experimental conditions, can produce cell death. Chemical analysis of spark-decomposed SF/sub 6/ has identified the major decomposition pathways and byproducts. Biological testing of individual byproduct mixtures has indicated that these major decomposition products may not account for the majority of the cell-killing effects seen in the assays. Further experiments have suggested that S/sub 2/F/sub 10/ may be produced and accumulate under the specific decomposition conditions and that this compound may be a major contributor to the observed cell lethality. It is concluded that testing of samples from commercial facilities and assays of decomposed gas after ameliorative treatments would both be appropriate investigations. >

Critical DNA target size model of ionizing radiation-induced mammalian cell death.

A new model of mammalian cell killing by ionizing radiation is presented. This model, termed the critical DNA target size model, postulates that DNA double-strand breakage is the critical radiation-induced lesion and that the dose-response for such breakage can be non-linear due to the action of a saturable chemical repair process. DNA double-strand breakage occurring within critical targets (proto-oncogene- or common fragile site-associated sequences) is postulated to initiate recombination events with undamaged sequences, leading to chromosomal aberrations. The subsequent loss of acentric fragments at mitosis is postulated to prevent the continuity of the genome and to produce cell death by the induction of chromatin structural changes. Experimental evidence contrary to other radiation action models is examined, and the hypotheses of the model are justified.

Hematoporphyrin derivative photocytotoxicity of human glioblastoma in cell culture.

The parameters of hematoporphyrin-derivative (HpD) photocytotoxicity of human glioma cells in cell culture were studied to determine the optimum wavelength and power density of light, to investigate the influence of tissue oxygenation, and to evaluate the role of singlet oxygen and free radicals in producing cell death. Cell survival curves demonstrated a relative killing efficiency of 12:1 for violet compared to red light. Eighty joules of red light were required to produce 100% cell kill at an HpD concentration of 10 micrograms/ml, a level of HpD that has been quantitated in biopsies from patients receiving HpD photoradiation therapy. No difference in cellular killing efficiency was observed for power densities of red light varying from 10 to 100 mW/cm2. Cytotoxicity was directly related to O2 tension from 12 to 490 torr with a slight but consistent increase in cell kill at O2 tensions from 7 to 12 torr. Cytotoxicity was effectively quenched by beta-carotene, whereas mannitol had no effect, indicating that cytotoxicity is probably mediated via a mechanism involving singlet oxygen. This information may serve as a basis for more effective application of HpD photoradiation therapy and for designing protocols to study the efficacy of such therapy.

Cytotoxicity of S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine in isolated rat kidney cells.

S-(1,2-Dichlorovinyl)glutathione (DCVG) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) produced time- and concentration-dependent cell death in isolated rat kidney proximal tubular cells. AT-125 blocked and glycylglycine potentiated DCVG toxicity, indicating that metabolism by gamma-glutamyltransferase is required. S-(1,2-Dichlorovinyl)-L-cysteinylglycine, a putative metabolite of DCVG, also produced cell death, which was prevented by 1,10-phenanthroline, phenylalanylglycine, and aminooxyacetic acid, inhibitors of aminopeptidase M, cysteinylglycine dipeptidase, and cysteine conjugate beta-lyase, respectively. Aminooxyacetic acid and probenecid protected against DCVC toxicity, indicating a role for metabolism by cysteine conjugate beta-lyase and organic anion transport, respectively. DCVC produced a small decrease in cellular glutathione concentrations and did not change cellular glutathione disulfide concentrations or initiate lipid peroxidation. DCVC caused a large decrease in cellular glutamate and ATP concentrations with a parallel decrease in the total adenine nucleotide pool; these changes were partially prevented by aminooxyacetic acid. Both DCVG and DCVC inhibited succinate-dependent oxygen consumption, but DCVC had no effect when glutamate + malate or ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine were the electron donors. DCVC inhibited mitochondrial, but not microsomal, Ca2+ sequestration. These alterations in mitochondrial function were partially prevented by inhibition of DCVG and DCVC metabolism and were strongly correlated with decreases in cell viability, indicating that mitochondria may be the primary targets of nephrotoxic cysteine S-conjugates.

Neuronal alterations in hippocampus following severe hypoglycaemia: a light microscopic and ultrastructural study in the rat.

Because they induce similar neuropathological changes (ischaemic cell change with microvacuolization), it has been suggested that ischaemia, status epilepticus and hypoglycaemia produce cell death by similar mechanisms, especially those resulting from intracellular calcium accumulation. We have recently demonstrated microvacuolation of neurons, mitochondrial swelling (the electron microscopic correlate of microvacuolization) and massive mitochondrial calcium sequestration (using the pyroantimonate technique) following ischaemia or status epilepticus. We therefore studied the selectively vulnerable neurons of rat hippocampus by light and electron microscopy (including the pyroantimonate technique) following 30 and 60 min of EEG isoelectricity resulting from insulin hypoglycaemia. The neuropathology at the light and EM level is unique and different from that following status epilepticus or ischaemia. The most constant finding is dark cell change of the granule cells at the tip of the dentate gyrus. In contrast to status epilepticus and ischaemia, hippocampal pyramidal neurons are far less frequently involved. Microvacuoles are rarely seen and, when present, their ultrastructural correlate is swollen Golgi apparatus, not dilated mitochondria. No intracellular calcium accumulation is demonstrable with pyroantimonate technique. Thus the cellular alterations produced by hypoglycaemia differ in character and distribution from those produced by anoxia-ischaemia. Mitochondrial calcium accumulation is not prominent in cell death from hypoglycaemia. Whether calcium toxicity plays another, subtler role in hypoglycaemic brain injury is unknown.

Differential effects of viral infection on islet and pituitary cell lines.

Although reovirus infection may lead to changes in endocrine function in vivo, little is known about the precise interaction of reovirus with endocrine cells. In this study we have examined the effects of reovirus infection on two types of endocrine cells, GH4C1 cells and RINm5F cells. Both type 1 reovirus and type 3 reovirus infect the two cells lines and appear to grow equally well. Viral replication occurred within the first 24 h following infection after which viral titers remained stable for 3 days. By 48-72 h after viral infection, substantial cytopathic effects were noted in RINm5F cells infected with both type 1 and type 3 reovirus. In GH4C1 cells, type 3 reovirus was most effective in producing cell death, and type 1 reovirus was significantly less cytotoxic despite a similar viral titer. Only type 1 reovirus caused a specific inhibition of overall protein and DNA synthesis, and this occurred only in the RINm5F cells. Over the time course studied, GH4C1 cells successfully infected with type 1 reovirus demonstrated no cytopathic effects, and only minimal alterations in cellular function were noted. Intracellular insulin content and insulin secretion, a "luxury function" of the RINm5F cells, were also surprisingly well maintained in the first 48 h after viral infection. In addition, virally infected cells were able to respond to glyceraldehyde, an insulin secretagogue, although the response appeared to be somewhat blunted compared to that of control cells. These results suggest that viral infection of endocrine cells results in specific alterations that depend on the nature of the infecting virus. In addition, the cellular environment of the host cell may be an important determinant in the outcome of viral infection.

Biophysical implications of bubble dynamics

Evidence is reviewed, from theory and experiment, that biological systems can be affected by ultrasound at low levels, if resonant gas bodies are present. In a suspension of cells or other particles a pulsating gas bubble causes the particles to migrate toward its surface via radiation force. This motion, in addition to acoustic microstreaming, transports particles into the bubble near-field where they are subjected to highly localized stress fields. In plant leaves containing gas-filled channels, the ultrasonic intensity required to produce cell death varies with frequency, showing minima in ranges corresponding roughly to calculated frequencies for resonance of the channels.

Defective interfering particles modulate VSV infection of dissociated neuron cultures

Infection of dissociated neuron cultures of mice with VSV and its defective particle DI-T was studied using fluorescent light microscopy as well as transmission and scanning electron microscopy. When cultures are infected with wild virus, VSV replicates selectively in neurons, producing cell death within 24–48 hr. Sensory and immature neurons express viral antigen most rapidly. Viral antigen and viral budding sites are detected along the neuron some and dendrites. When large amounts of DI-T particles are added to the wild virus inoculum, viral growth is completely suppressed in mature neurons, the cell killing effects of VSV are considerably delayed and co-infected cultures survive for 5–16 days. Viral antigen accumulates in cytoplasmic inclusions and on the membrane of neuron cell somas and dendrites in the virtual absence of viral assembly. Identical modulation of VSV infection in mature neuron cultures is obtained when DI-T particles are added before or after the wild virus, but ultraviolet inactivation of DIs completely abolishes their protective effect. Immature neurons or Vero cells cannot be protected from acute cytopathic changes by an equivalent amount of DI particles. Thus DIs interfere with replication and assembly of the wild virus and attenuate cell killing effects in mature neurons in vitro.

Destruction of the sympathetic nervous system in neonatal rats and hamsters by vinblastine: prevention by concomitant administration of nerve growth factor

If the primary mechanism by which nerve growth factor (NGF) gains access to the cell body is a specific uptake and retrograde transport from the periphery, then an inhibitor of axoplasmic transport would be expected to produce cell death. Such an inhibitor was administered to neonatal and adult rats and hamsters. A single injection of vinblastine (0.25–0.5 mg/kg s.c.) to 2-day-old rats produced massive cell death in the superior cervical ganglia (SCG). The levels of tyrosine hydroxylase in the SCG were reduced 80% at doses of 0.4 mg/kg, a dose which was uniformly fatal. Doses of 0.25 mg/kg at two days of age resulted in a 50% mortality rate and the survivors showed a permanent, partial sympathectomy. The adrenal medulla and sensory neurons did not appear to be affected. Administration of a single injection of vinblastine at maximally tolerated doses did not destroy sympathetic neurons in animals 14 days of age or older. Chronic administration of vinblastine (0.5 mg/kg every other day for two weeks) to adult rats did not destroy sympathetic neurons. Similar results were obtained in hamsters. Concomitant administration of NGF appeared to completely prevent the cytotoxic effects of vinblastine assessed by both morphological and enzymatic criteria. Several explanations are discussed which might explain these results. The similarities in the effects of vinblastine, other drugs, and axonal crush on sympathetic neurons are noted. It is proposed that all drugs which destroy sympathetic neurons (guanethidine, 6-hydroxydopamine, vinblastine) as well as anti-NGF and axonal ligation destroy the neurons by depriving the cell body of NGF from the periphery.

Purification of a cytotoxic factor from human and rat tissues.

Human and rat normal tissues and tumours have been studied for the presence of toxic substances, possibly of importance in the development of cachexia in patients with cancer and other chronic diseases. The toxic effect of tissue extracts was gauged by measuring the inhibition of growth of mouse L-cells in 1-ml cultures, as revealed by reduced incorporation of [14C]leucine into cell protein. A common cytotoxic substance of mol. wt. approximately 700 daltons was isolated from all rat and human tissues tested, including tumours. The isolation procedure involved tissue homogenization, followed by pressure dialysis, gel filtration of concentrated pressure dialysates, cation exchange chromatography, and thin layer chromatography. Amounts isolated from different tissues varied by a factor of 3. The purified substance reacted with ninhydrin and a few other reagents for amino groups. It was completely resistant to acid and enzymatic hydrolysis. The evidence thus suggests that the substance is an amine. It is toxic to L-cells, HeLa cells and normal rat fibroblasts in concentrations of 10-20 muM, producing cell death and lysis during incubation overnight.

Role of cyclic amp in differentiation of human neuroblastoma cells in culture

The inhibitors of cyclic AMP phosphodiesterase (papaverine and 4-(-3-butoxy-4-methoxybenzyl)-2-imidazolidinone), serum-free medium, and x irradiation caused cell death and neurite formation in human neuroblastoma cells in culture (IMR-32), whereas theophylline was ineffective. Prostaglandin (PG) E1, N6O'2-dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) induced neurites without causing cell lethality. Inhibitors of phosphodiesterase and PGE1 increased the intracellular level of cAMP by about 2- and 4-fold respectively, whereas serum-free medium and x irradiation did not. The combination of PGE1 and phosphodiesterase inhibitor was more effective in causing morphological differentiation and in increasing the cAMP level than the individual agent. Sodium butyrate induced cell death and neurites, probably in part by increasing the cAMP level. cAMP, guanosine 3',5'-cyclic monophosphate, and adenosine had no detectable effect on the growth or morphology of neuroblastoma cells in culture. Adenosine 5'-monophosphate produced cell death without causing neurite formation. DbcAMP, and to a much lesser degree, sodium butyrate increased the tyrosine hydroxylase activity.