The Experts below are selected from a list of 231 Experts worldwide ranked by ideXlab platform

Gary E. Isom - One of the best experts on this subject based on the ideXlab platform.

  • Cyanide‐Induced Apoptosis and Oxidative Stress in Differentiated PC12 Cells
    Journal of neurochemistry, 2002
    Co-Authors: Edward M. Mills, Goran Pavlaković, Palur G. Gunasekar, Gary E. Isom
    Abstract:

    Terminally differentiated PC12 cells are a useful neuron-like model for studying programmed cell death in response to nerve growth factor (NGF) deprivation. This in vitro model was used to investigate the mechanism by which cyanide-induced Histotoxic Hypoxia produces neuronal degeneration. Treatment of undifferentiated PC12 cells with 0.1 mM KCN for 24 h did not produce cell death. In contrast, treatment of differentiated PC12 cell cultures with 0.1 mM KCN for 24 h increased cell death by 43% when compared with control cultures, as measured by trypan blue dye exclusion and lactate dehydrogenase release assays. The Ca2+/Mg(2+)-dependent endonuclease inhibitor aurintricarboxylic acid and the transcriptional inhibitor actinomycin D partially attenuated hypoxic toxicity, suggesting roles for endonuclease activation and transcription in this model of neuronal death. Extracted DNA from cyanide-treated neurons demonstrated cleavage into oligonucleosomal fragments on gel electrophoresis. Transmission electron microscopic analysis showed morphological changes consistent with apoptotic cell death, including membrane blebbing and convolution, as well as chromatin condensation and margination to the nuclear membrane. Addition of either ascorbate or catalase to the cultures partially attenuated the loss of cell viability induced by cyanide, and decreased the incidence of apoptotic cells after treatment, based on the in situ detection of DNA strand breaks. The ability of cyanide to elevate intracellular oxidant species was determined by microfluorescence in differentiated PC12 cells loaded with the oxidant-sensitive dye 2',7'-dichlorofluorescin. Exposure of cells to 0.1 mM KCN produced a rapid generation of oxidants that was blocked approximately 50% by ascorbate or catalase. These observations indicate that cyanide induces apoptosis in terminally differentiated, and not undifferentiated, PC12 cells, and that antioxidants significantly reduce the incidence of cyanide-induced apoptosis.

  • Cyanide induces protein kinase C translocation: Blockade by NMDA antagonists
    Journal of biochemical toxicology, 1994
    Co-Authors: A. Rathinavelu, Peiwen Sun, Goran Pavlaković, Joseph L. Borowitz, Gary E. Isom
    Abstract:

    Activation and translocation of protein kinase C (PKC) during KCN-induced Histotoxic Hypoxia was studied in rat brain slices prepared from cerebellum, hippocampus, and cortex. Treatment with 1-10 mM KCN produced a significant increase in PKC translocation and enzyme activity in the particulate fraction of cerebellar and hippocampal slices. In cortical slices, PKC activity was not affected by cyanide treatment. The membrane-associated PKC activity reached a maximum 30 minutes after incubation with KCN and remained elevated up to 60 minutes in both the hippocampus and cerebellum. Pretreatment with MK-801 and APV, specific NMDA receptor antagonists, blocked the cyanide-stimulated translocation in the hippocampus and cerebellum, whereas CNQX, an AMPA/kainate receptor antagonist, did not alter the response. These results demonstrate that cyanide stimulates PKC activation and translocation from the cytosol to membranes in select brain areas and NMDA receptor activation mediates this process.

  • Activation of a Calcium- and pH-Dependent Phospholipase A2 by Cyanide in PC12 Cells
    Toxicology and applied pharmacology, 1994
    Co-Authors: Ching-wen Yang, Joseph L. Borowitz, A. Rathinavelu, Gary E. Isom
    Abstract:

    Abstract Previous studies suggested that alterations in phospholipid composition of plasma membranes may contribute to neuronal injury associated with cyanide-induced Histotoxic Hypoxia. This prompted a study of the effects of KCN on phospholipase A2 (PLA2), an enzyme which catalyzes breakdown of membrane phospholipids. PLA2 activity was measured by quantitating the release of [3H]arachidonic acid ([3H]AA) from rat pheochromocytoma (PC12) cells. KCN produced a time (1-15 min)- and concentration (0.5-10 mM)-dependent release of [3H]AA from the cells. When cells were incubated in Ca2+-free buffer, KCN (5 mM) was still able to release [3H]AA. In cells loaded with BAPTA, an intracellular Ca2+ chelator, cyanide-induced release of [3H]AA was blocked, indicating that mobilization of intracellular Ca2+ can activate the enzyme. The PLA2 inhibitors dibucaine (50 μM) and mepacrine (50 μM) inhibited KCN-mediated [3H]AA release. Incubation of PC12 cells in an extracellular pH of 6.50 reduced the KCN effect, whereas incubation at pH 7.90 enhanced [3H]AA release. These data indicate that in PC12 cells KCN activates a Ca2+- and pH-dependent PLA2 which may contribute to cyanide-induced cell damage.

  • Cyanide induces Ca2+-dependent and -independent release of glutamate from mouse brain slices
    Neuroscience letters, 1991
    Co-Authors: Manisha N. Patel, Barbara K. Ardelt, George K.w. Yim, Gary E. Isom
    Abstract:

    The effect of cyanide-induced Histotoxic Hypoxia on endogenous glutamate release from mouse cortical, cerebellar and hippocampal slices was studied. Incubation of slices with cyanide over a 30 min period resulted in extracellular accumulation of glutamate which was decreased in the absence of Ca2+ in the incubation media. When glutamate release was continuously monitored by fluorometry, cyanide initiated a rapid release of glutamate. This initial release was found to be independent of extracellular calcium. Depolarizing concentrations of potassium chloride produced a predominantly Ca(2+)-dependent release. It is concluded that cyanide exposure induced a rapid release of endogenous glutamate mediated by both Ca(2+)-dependent and Ca(2+)-independent mechanisms.

  • Cyanide-induced alteration of the adenylate energy pool in a rat neurosecretory cell line.
    Journal of applied toxicology : JAT, 1991
    Co-Authors: Edward U. Maduh, Joseph L. Borowitz, Gary E. Isom
    Abstract:

    Cultures of a rat PC12 pheochromocytoma neurosecretory cell line were used to determine the responsiveness of oxidative energy status of isolated neuronal cells to cyanide exposure. Intracellular levels of ATP and its immediate metabolites, ADP and AMP, were measured in monolayer cultures of PC12 cells incubated for 0–30 min with KCN (10 mM). Over the period 2.5–30 min, cyanide treatment decreased ATP levels by 32–51% but ADP and AMP levels were not altered significantly. Additionally, ATP/ADP and ATP/AMP ratios were significantly reduced in KCN-intoxicated cells. These alterations in energy status may explain the prompt ablation of ion homeostasis reported previously in this model upon exposure to KCN. The energy-depleting actions of cyanide were not modified by pretreatment of cells with diltiazem, a calcium channel antagonist demonstrated to possess cytoprotective activity against Histotoxic Hypoxia induced by cyanide. Since PC12 cells rapidly respond to cyanide, with predictable depletions of the cell adenylate energy pool, this cell line can serve as a suitable in vitro model for studies of neurotoxicity involving ischemic/hypoxic conditions.

D F Donnelly - One of the best experts on this subject based on the ideXlab platform.

  • Response to cyanide of two types of glomoid cells in mature rat carotid body.
    Brain research, 1993
    Co-Authors: D F Donnelly
    Abstract:

    Cells belonging to glomoids of mature rat carotid bodies were studied using the whole-cell patch clamp technique following acute dissociation. The recorded population encompassed two subtypes: one type (n = 202), termed G(out), was characterized by a small voltage-dependent inward current (43 +/- 9 pA, mean +/- S.E.M.), large outward current (671 +/- 31 pA @ +40 mV), high membrane resistance (1910 +/- 110 M omega) and low capacitance (5.1 +/- 0.1 pF). A second subtype (n = 56), termed G(in), had significantly lower membrane resistance (177 +/- 35 M omega), higher membrane capacitance (15.0 +/- 1.0 pF) and little voltage-dependent current. Neither subtype supported generation of multiple action potentials during depolarization in the current clamp mode. Intracellular staining of the recorded cells by Lucifer yellow showed co-localization of both subtypes to clusters of cells which stained positively for catecholamines. Somal diameter was slightly, but significantly, larger for G(in) cells (8.7 +/- 0.4 microM, n = 7) compared to G(out) cells (7.8 +/- 0.2 microM, n = 31) and all cells had fine cytoplasmic processes extending around neighboring cells. During recordings using the perforated patch technique, Histotoxic Hypoxia significantly decreased a voltage-dependent outward current in G(out) cells by 113 +/- 60 pA (n = 13), and decreased the holding current by 10 +/- 4 pA (n = 13) from a control value of -32 +/- 6 pA. In G(in) cells, cyanide significant decreased membrane resistance and decreased holding current by 55 +/- 28 pA from a control value of +120 +/- 42 pA (n = 7), but caused no significant change in outward current. These results show that glomoids of mature rat carotid bodies contain at least two types of cells which differ in their morphologic and electrophysiologic characteristics. The subtypes rapidly respond to Histotoxic Hypoxia and thus may mediate separate roles in the organ response to chemostimuli.

  • Response to cyanide of two types of glomoid cells in mature rat carotid body
    Brain Research, 1993
    Co-Authors: D F Donnelly
    Abstract:

    Cells belonging to glomoids of mature rat carotid bodies were studied using the whole-cell patch clamp technique following acute dissociation. The recorded population encompassed two subtypes: one type (n=202), termed G(out), was characterized by a small voltage-dependent inward current (43±9pA, mean ±S.E.M.), large outward current (671±31 pA@+40 mV), high membrane resistance (1910 ± 110M Ω) and low capacitance (5.1 ± 0.1pF). A second subtype (n=56), termed G(in), had significantly lower membrane resistance (177 ± 35 MΩ), membrane capacitance (15.0 ± 1.0 pF) and little voltage-dependent current. Neither subtype supported generation of multiple action potentials during depolarization in the current clamp mode. Intracellular staining of the recorded cells by Lucifer yellow showed co-localization of both subtypes to clusters of cells which stained positively for catecholamines. Somal diameter was slightly, but significantly, larger for G(in) cells 8.7 ± 0.4 μM, n=7) compared to G(out) cells (7.8±0.2 μM, n=31) and all cells had fine cytoplasmic process s extending around neighboring cells. During recordings using the perforated patch technique, Histotoxic Hypoxia significantly decreased a voltage-dependent outward current in G(out) cells by 113±60pA (n=13), and decreased the holding current by 10±4pA (n=13) from a control value of −32±6pA. In G(in) cells, cyanide significant decreased membrane resistance and decreased holding current by 55±28pA from a control value of +120±42pA (n=7), but caused no significant change in outward current. These results show that glomoids of mature rat carotid bodies contain at least two types of cells which differ in their morphologic and electrophysiologic characteristics. The subtypes rapidly respond to Histotoxic Hypoxia and thus may mediate separate roles in the organ response to chemostimuli.

  • Electrochemical detection of catecholamine release from rat carotid body in vitro
    Journal of applied physiology (Bethesda Md. : 1985), 1993
    Co-Authors: D F Donnelly
    Abstract:

    Neurotransmitter secretion from carotid body glomus cells is hypothesized to be an essential element of chemotransduction. To address one aspect of this hypothesis, catecholamine release in response to hypoxic Hypoxia and Histotoxic Hypoxia was examined using electrically treated carbon-fiber microelectrodes placed in rat carotid bodies in vitro. Carotid bodies of mature rats were removed, along with a portion of the sinus nerve, and suspended in oxygenated (95% O2–5% CO2) Ringer saline at 35 degrees C. The microelectrode differential current after a 50-mV step was recorded over the potential range of -300 to +500 mV. In some preparations, a suction electrode applied to the sinus nerve recorded single-fiber chemoreceptor afferent activity. Stimulation by severe Hypoxia (Po2 approximately 0–10 Torr for 3 min, n = 10) and cyanide (2 mM for 2 min) caused an increase in sinus nerve activity and an increase in the carbon-fiber electrode current at a potential corresponding to the oxidation potential of dopamin...

R. Bhattacharya - One of the best experts on this subject based on the ideXlab platform.

  • Oxidative stress mediated cytotoxicity of cyanide in LLC-MK2 cells and its attenuation by alpha-ketoglutarate and N-acetyl cysteine.
    Toxicology letters, 2008
    Co-Authors: J. Hariharakrishnan, Ravindra M. Satpute, G.b.k.s. Prasad, R. Bhattacharya
    Abstract:

    Cyanide is a rapidly acting mitochondrial poison that inhibits cellular respiration and energy metabolism leading to Histotoxic Hypoxia followed by cell death. Cyanide is predominantly a neurotoxin but its toxic manifestations in non-neuronal cells are also documented. This study addresses the oxidative stress mediated cytotoxicity of cyanide in Rhesus monkey kidney epithelial cells (LLC-MK2). Cells were treated with various concentrations of potassium cyanide (KCN) for different time intervals and cytotoxicity was evidenced by increased leakage of intracellular lactate dehydrogenase, mitochondrial dysfunction (MTT assay) and depleted energy status of cells (ATP assay). Cytotoxicity was accompanied by lipid peroxidation indicated by elevated levels of malondialdehyde (MDA), reactive oxygen species (ROS) and reactive nitrogen species (RNS) (DCF-DA staining), diminished cellular antioxidant status (reduced glutathione (GSH), glutathione peroxidase, superoxide dismutase and catalase). These cascading events triggered an apoptotic kind of cell death characterized by oligonucleosomal DNA fragmentation and nuclear fragmentation (Hoechst 33342 staining). Apoptosis was further confirmed by increased caspase-3 activity. Cyanide-induced cytotoxicity, oxidative stress, and DNA fragmentation were prevented by alpha-ketoglutarate (A-KG) and N-acetyl cysteine (NAC). A-KG is a potential cyanide antidote that confers protection by interacting with cyanide to form cyanohydrin complex while NAC is a free radical scavenger and enhances the cellular GSH levels. The study reveals cytotoxicity of cyanide in cells of renal origin and the protective efficacy of A-KG and NAC.

  • Pharmacological interventions of cyanide-induced cytotoxicity and DNA damage in isolated rat thymocytes and their protective efficacy in vivo.
    Toxicology letters, 2001
    Co-Authors: R. Bhattacharya, P.v. Lakshmana Rao
    Abstract:

    Abstract Cyanide inhibits the mitochondrial respiratory chain enzyme cytochrome oxidase causing Histotoxic Hypoxia. It is primarily considered as a neurotoxin but its other toxic manifestations are also well documented. Cyanide-induced apoptosis in neuronal cells has also been demonstrated recently. At the same time we also reported that potassium cyanide (KCN) produces extensive cytotoxicity and DNA fragmentation in rat thymocytes. The DNA damage was sensitive to elevated levels of extracellular Ca 2+ and was attenuated by Zn 2+ (modulator of Ca 2+ -dependent endonuclease), N -acetylcysteine (free radical scavenger) and diltiazem (Ca 2+ channel blocker). In a continuation of this work, in the present study we have shown that the cytotoxicity and DNA fragmentation induced by 5 mM KCN was preceded by loss of mitochondrial integrity (MTT assay and rhodamine-123 staining) and nuclear viability (propidium iodide uptake) which were mediated by generation of reactive oxygen species (DCHF-DA staining). The DNA damage was also accompanied by nuclear fragmentation (Hoechst 33342 staining), a phenomenon that characterises the ‘apoptotic’ type of cell death. The in vitro toxic insult of KCN was challenged by pre-treatment (0.5 h), simultaneous treatment or post-treatment (0.5–3 h) of various pharmacological agents viz., Trolox® (antioxidant), EGTA (Ca 2+ modulator) and aurintricarboxylic acid (ATA; Ca 2+ /Mg 2+ -dependent endonuclease inhibitor). In addition, Quercetin (antioxidant) was tested as simultaneous treatment alone and was found to be ineffective. On the basis of various biochemical indices and DNA fragmentation (quantitative and qualitative), simultaneous treatment of Trolox® was found to be the most effective in attenuating cyanide toxicity in vitro. This protection can be attributed to interventions in oxidative stress-mediated cell injury which is an early event preceding DNA damage. Both EGTA and ATA could not prevent this damage. Trolox® also increased the LD 50 of KCN in mice 2.5-fold as compared to 1.8- and 1.6-fold for EGTA and ATA, respectively.

  • Cyanide induced DNA fragmentation in mammalian cell cultures
    Toxicology, 1997
    Co-Authors: R. Bhattacharya, P.v. Lakshmana Rao
    Abstract:

    Cyanide is a mitochondrial poison and its toxicity is mediated through Histotoxic Hypoxia. Although cyanide is regarded as a neurotoxin, its other toxic manifestations are also well documented. Cyanide triggers all those events which can lead to DNA damage, but its genotoxic potential has not been established yet. The present investigation addresses the DNA damage induced by cyanide in rat thymocytes in vitro. Cell viability (eosin Y exclusion and LDH leakage) along with DNA strand breaks were measured in thymocytes exposed to 1.25–10 mM KCN for various time intervals. Cleavage into oligonucleosomal fragments of extracted DNA from cyanide treated thymocytes were visualized on gel electrophoresis. Cyanide produced both time and dose dependent DNA fragmentation accompanied by cytotoxicity. The DNA damage was sensitive to elevated levels of extracellular Ca2+ and was minimal in Ca2+ free medium. The DNA fragmentation was attenuated by Zn2+ (modulator of Ca2+/Mg2+-dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca2+ channel blocker). Cyanide induced DNA damage was further observed in baby hamster kidney cells (BHK-21), where unlike thymocytes, internucleosomal DNA fragmentation was not observed. Thymocytes were more sensitive to cyanide as compared to BHK-21 cells.

P.v. Lakshmana Rao - One of the best experts on this subject based on the ideXlab platform.

  • Pharmacological interventions of cyanide-induced cytotoxicity and DNA damage in isolated rat thymocytes and their protective efficacy in vivo.
    Toxicology letters, 2001
    Co-Authors: R. Bhattacharya, P.v. Lakshmana Rao
    Abstract:

    Abstract Cyanide inhibits the mitochondrial respiratory chain enzyme cytochrome oxidase causing Histotoxic Hypoxia. It is primarily considered as a neurotoxin but its other toxic manifestations are also well documented. Cyanide-induced apoptosis in neuronal cells has also been demonstrated recently. At the same time we also reported that potassium cyanide (KCN) produces extensive cytotoxicity and DNA fragmentation in rat thymocytes. The DNA damage was sensitive to elevated levels of extracellular Ca 2+ and was attenuated by Zn 2+ (modulator of Ca 2+ -dependent endonuclease), N -acetylcysteine (free radical scavenger) and diltiazem (Ca 2+ channel blocker). In a continuation of this work, in the present study we have shown that the cytotoxicity and DNA fragmentation induced by 5 mM KCN was preceded by loss of mitochondrial integrity (MTT assay and rhodamine-123 staining) and nuclear viability (propidium iodide uptake) which were mediated by generation of reactive oxygen species (DCHF-DA staining). The DNA damage was also accompanied by nuclear fragmentation (Hoechst 33342 staining), a phenomenon that characterises the ‘apoptotic’ type of cell death. The in vitro toxic insult of KCN was challenged by pre-treatment (0.5 h), simultaneous treatment or post-treatment (0.5–3 h) of various pharmacological agents viz., Trolox® (antioxidant), EGTA (Ca 2+ modulator) and aurintricarboxylic acid (ATA; Ca 2+ /Mg 2+ -dependent endonuclease inhibitor). In addition, Quercetin (antioxidant) was tested as simultaneous treatment alone and was found to be ineffective. On the basis of various biochemical indices and DNA fragmentation (quantitative and qualitative), simultaneous treatment of Trolox® was found to be the most effective in attenuating cyanide toxicity in vitro. This protection can be attributed to interventions in oxidative stress-mediated cell injury which is an early event preceding DNA damage. Both EGTA and ATA could not prevent this damage. Trolox® also increased the LD 50 of KCN in mice 2.5-fold as compared to 1.8- and 1.6-fold for EGTA and ATA, respectively.

  • Cyanide induced DNA fragmentation in mammalian cell cultures
    Toxicology, 1997
    Co-Authors: R. Bhattacharya, P.v. Lakshmana Rao
    Abstract:

    Cyanide is a mitochondrial poison and its toxicity is mediated through Histotoxic Hypoxia. Although cyanide is regarded as a neurotoxin, its other toxic manifestations are also well documented. Cyanide triggers all those events which can lead to DNA damage, but its genotoxic potential has not been established yet. The present investigation addresses the DNA damage induced by cyanide in rat thymocytes in vitro. Cell viability (eosin Y exclusion and LDH leakage) along with DNA strand breaks were measured in thymocytes exposed to 1.25–10 mM KCN for various time intervals. Cleavage into oligonucleosomal fragments of extracted DNA from cyanide treated thymocytes were visualized on gel electrophoresis. Cyanide produced both time and dose dependent DNA fragmentation accompanied by cytotoxicity. The DNA damage was sensitive to elevated levels of extracellular Ca2+ and was minimal in Ca2+ free medium. The DNA fragmentation was attenuated by Zn2+ (modulator of Ca2+/Mg2+-dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca2+ channel blocker). Cyanide induced DNA damage was further observed in baby hamster kidney cells (BHK-21), where unlike thymocytes, internucleosomal DNA fragmentation was not observed. Thymocytes were more sensitive to cyanide as compared to BHK-21 cells.

Jörg B. Schulz - One of the best experts on this subject based on the ideXlab platform.

  • Extended therapeutic window for caspase inhibition and synergy with MK-801 in the treatment of cerebral Histotoxic Hypoxia
    Cell Death & Differentiation, 1998
    Co-Authors: Jörg B. Schulz, Russell T. Matthews, Michael Weller, Michael T Heneka, Peter Groscurth, Jean-claude Martinou, Jürgen Lommatzsch, Rainer Von Coelln, Ullrich Wüllner, Peter-a Löschmann
    Abstract:

    In rats, striatal Histotoxic hypoxic lesions produced by the mitochondrial toxin malonate resemble those of focal cerebral ischemia. Intrastriatal injections of malonate induced cleavage of caspase-2 beginning at 6 h, and caspase-3-like activity as identified by DEVD biotin affinity-labeling within 12 h. DEVD affinity-labeling was prevented and lesion volume reduced in transgenic mice overexpressing BCL-2 in neuronal cells. Intrastriatal injection of the tripeptide, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a caspase inhibitor, at 3 h, 6 h, or 9 h after malonate injections reduced the lesion volume produced by malonate. A combination of pretreatment with the NMDA antagonist, dizocilpine (MK-801), and delayed treatment with zVAD-fmk provided synergistic protection compared with either treatment alone and extended the therapeutic window for caspase inhibition to 12 h. Treatment with cycloheximide and zVAD-fmk, but not with MK-801, blocked the malonate-induced cleavage of caspase-2. NMDA injections alone resulted in a weak caspase-2 cleavage. These results suggest that malonate toxicity induces neuronal death by more than one pathway. They strongly implicate early excitotoxicity and delayed caspase activation in neuronal loss after focal ischemic lesions and offer a new strategy for the treatment of stroke.

  • Improved Therapeutic Window for Treatment of Histotoxic Hypoxia with a Free Radical Spin Trap
    Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 1995
    Co-Authors: Jörg B. Schulz, Russell T. Matthews, Bruce G. Jenkins, Preetinder Brar, M. Flint Beal
    Abstract:

    The therapeutic time window for N-methyl-d-aspartate (NMDA) antagonists, non-NMDA antagonists, and glutamate release inhibitors in focal models of ischemia appears to be about 1–2 h. In contrast, a free radical spin trap was found to have an improved therapeutic window. We compared the therapeutic time windows of the NMDA antagonist dizolcilpine maleate (MK-801), the glutamate release inhibitor lamotrigine, and the free radical spin trap n-tert-butyl-α-(2-sulfophenyl)-nitrone (S-PBN) against striatal lesions produced by the mitochondrial toxin malonate, which produces Histotoxic Hypoxia. Lamotrigine exerted neuroprotective effects when administered at 1 h before malonate injections. MK-801 protected at 1 h before and 1 h after malonate injections, whereas S-PBN showed efficacy when administered up to 6 h after malonate injections. Striatal injections of malonate produced a rapid increase in lactate production and early changes in diffusion-weighted imaging as assessed by magnetic resonance imaging. Theref...