Javier Guerrero, Ubaldo Soto, Soledad Miranda, Rocio Foncea, and Federico Leighton
Laboratorio de Citología Bioquímica y Lípidos, Facultad de Ciencias Biológicas, Pontificia Universidad. Católica de Chile, Casilla 114-D, Santiago, Chile
Mitochondria are responsible for the generation of a large fraction of the free radicals in the cell. Leakage of electrons from the respiratory chain leads to reactive oxygen species (ROS) instead of H2O. Experimental evidences suggest that mitochondrial ROS generation increases in aging or otherwise damaged mitochondria; and it also increases in conditions of augmented energy demand as, for example, in contracting muscle cells. Mitochondrial biogenesis is the result of the coordinated expression of nuclear and mitochondrial genes. Mitochondrial transcription factor A (mtTFA) is codified in the nucleus and regulates transcription in mitochondria. In the nucleus, the nuclear respiratory factor 1 (NRF-1) another transcription factor, regulates the expression of mtTFA and other mt genes.
In this work, we propose and explore the hypothesis that mitochondrial signaling to the nucleus, for the coordinated regulation of the mitochondrial genes encoded both, in the nucleus and in the mitochondria, is mediated by ROS.
To evaluate this hypothesis, we studied the expression of NRF-1, mtTFA and CoxIII (a subunit of cytochrome oxidase) in HeLa cells exposed to 100 µM menadione. The mRNAs for these three genes were detected by semiquantitative RT-PCR, since the expression level of the transcription factors is low. A temporal pattern of induction was observed, beginning with NRF-1 and mtTFA (0.5-2 h) followed by CoxIII (4 h). Similar but much weaker results were observed with H2O2 instead of menadione. These findings support the proposed role of ROS in mitochondrial signaling to the nuclear mitochondrial genes.
Supported by FONDECYT 1960637, and the Molecular Basis of Chronic Diseases Program, PUC, research projects
An oxidative stress model of glutamate cytotoxicity in HT-4 hippocampal cells
Derick Han*, Chandan K. Sen, Michael S. Kobayashi, Sashwati Roy, Enrique Cadenas*, and Lester Packer
*Department of Molecular Pharmacology & Toxicologyf, School of Pharmacy, University of Southern California, Los Angeles, and Department of Molecular and Cell Biology, University of California, Berkeley, CA , USA
The addition of exogenous glutamate to primary cultured neurons elicits a rapid cell death. Glutamate cytotoxicity is believed to be mediated through an overstimulation of the NMDA receptor and has therefore been termed ³excitotoxicity². However, glutamate has also been shown to be cytotoxic in neuronal cell (immature cortical neurons, PC12, C6, HT-4) that lack the NMDA receptors. This type of glutamate cytotoxicity has been primarily attributed to a loss of intracellular glutathione (GSH). Cystine transport, needed for GSH synthesis, has been shown to be inhibited by glutamate, resulting in a rapid loss of intracellular GSH. GSH-depleted cells are believed to die from oxidative stress, although the exact mechanism remains unclear. Antioxidants like vitamin E and lipoic acid have been shown to protect cells from glutamate cytotoxicity. Using HT-4 cells, we demonstrate that glutamate induced cytotoxicity is not solely due to a depletion of intracellular GSH, but also involves activation of a metabotrophic glutamate receptor. Metabotrophic receptors are G protein linked receptors that regulate a wide range of secondary messengers. Treatment of HT-4 cells with quisqualic acid, a metabo trophic receptor agonist stronger than glutamate, results in a rapid cell death that could be inhibited by vitamin E. MCPG, a potent competitive antagonist of the metabotrophic glutamate receptor, prevented glutamate-induced cell death. The glutamate activation of the metabotrophic receptor likely upregulates an oxidase involved in cytotoxicity. Increased peroxide generation observed after glutamate treatment could be inhibited by treatment of cells with cycloheximide, a protein synthesis inhibitor. Thus, we suggest that non-NMDA mediated glutamate cytotoxicity involves two mechanisms: a glutamate inhibition of cystine transport to lower GSH levels and a glutamate activation of a metabotrophic receptor that upregulates an oxidase. Glutamate induced cytotoxicity is then an end result of the cells inability, due to GSH depletion, to deal with a glutamate induced increase in the level of oxidants.
Han D, Sen CK, Roy S, Kobayashi MS, Tritschler H, Packer L (1997) Am. J. Physiol. 273, R1771-R1778
Th. Herrling, J. Fuchs*, and N. Groth#
FOM - University of Applied Science and Technology Berlin, *Johann-Wolfgang Goethe University Frankfurt, #Galenus Inc. Berlin
Biological systems are a potential target of oxidative injury because they are continuously exposed to a high oxygen concentration and contains a variety of oxydizible structures critical for maintenance of cellular homeostasis. Each biological cell has an antioxygenic potential which is determined by the balance between factors promoting antioxidant and those exciting antioxidant action. Oxidative damage in lipids, proteins and nucleic acids can be repaired by antioxidants. The effectiveness of this prevention, interception and repair mechanisms is determined by the Antioxidative Potential (AOP). We have developed an assay for the free radical-reducing activity of antioxidants and biological tissues that is based on the one-electron reduction of the nitroxide radical Tempol (1) and the stable free radical DPPH (2). This substances are relatively persistent radicals whose concentration can easily be quantitated by ESR spectroscopy. The both assays had been applied to different antioxidants and different biological tissues. On the basis of the measurements we have defined the Radical Protection Factor (RPF) which determines the reduction potential of the probes investigated. The RPF is a normalized factor which enables the comparison between different antioxidant substances (vitamins) and biological tissues. Diagrams based on the RPF's of this different probes are represented and discussed.
J.Fuchs, R. Mehlhorn, L. Packer; Assay for Free Radical Reductase Activity in Biological Tissue by Electron Spin Resonance Spectroscopy. Methods in Enzymol. 186, 670-674; 1990
M.S. Blois; Antioxidant Determinations by the Use of a Stable Free Radical. Nature. 181; 1199-1200; 1958
E.Ho, G. Chen and T.M. Bray
The Ohio State University, Columbus, OH 43210
IDDM is a complex, multifactorial disease characterized by profound pancreatic b cell destruction. The cellular mechanisms leading to b cell death are still unclear. However, there is growing evidence that free radicals are involved in the pathway leading to the development of IDDM. We hypothesize that free radical production and the activation of NF-kB, an oxidative stress responsive transcription factor, are key signals in initiating the cascade of events leading to b cell death and IDDM. PBN is a commonly used spin-trapping agent that may inhibit these processes and inhibit the development of IDDM. The primary goal of the study was to determine the efficacy of PBN in preventing alloxan-induced diabetes. Weanling CD1 mice were administered with PBN (150 mg/kg BW, i.p.) and then challenged (i.p.) with 0, 150, 200 or 300 mg/kg BW alloxan. Fasting blood glucose levels were monitored for 9 days. At all doses, PBN significantly reduced alloxan-induced hyperglycemia. To elucidate mechanisms by which PBN prevents hyperglycemia, we investigated the ability of PBN a) to trap alloxan-induced radicals with electron paramagnetic resonance (EPR) and b) to inhibit the activation of NF-kB by alloxan using electrophoretic mobility shift assay (EMSA). EPR studies using PBN, provided direct evidence that alloxan produces free radicals and PBN can directly trap these radicals, thus reducing their reactivity. At the same time, PBN inhibited alloxan-induced NF-kB activation. In addition, PBN reduced nitric oxide production and elevated supero xide dismutase (SOD) activity in the pancreas. This suggests that the protective effect of PBN in alloxan-induced hyperglycemia may be attributed to the stabilization of free radical species and the prevention ROS-induced NF-kB activation. Inhibition of these early signals in the diabetogenic pathway halts the cascade events leading to b cell death and the development of IDDM.
Katsuki Imao, H. Wang*, M. Komatsu** and M. Hiramatsu**
SAIDO Co., 2-13 3-chome, Befu, Jyonan-ku Fukuoka 814-0104, *Department of 1st Biochemistry, Yamagata University, School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-2331 and **Institute for Life Support Technology, Yamagata Technopolis Foundation, 2-2-1 Matsuei, Yamagata 990-2473, Japan
Fermented papaya preparation is a natural health food that has been commercially sold in Japan for 2 years. It is made by yeast fermentation of Carica Papaya Linn. We examined the antioxidant action of the fermented papaya preparation on free radicals and lipid peroxidation. Free radicals have been related with aging and diseases, such as cancer, diabetes and especially in neurological disorders, for example, Parkinson's disease or Alzheimer's disease. A diet including variable antioxidant foods may therefore help to prevent these illnesses.
The free radical scavenging activity of the fermented papaya preparation was examined using an electron spin resonance (ESR) spectrometer. Fermented papaya preparation (50mg/ml) scavenged 80% of hydroxyl radicals (.OH) as spin adducts of spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (5.27 x 1015 spins/ml) generated by Fenton reagents. The value of IC50 was 12.5mg/ml. The oral administration of the fermented papaya preparation for 4 weeks decreased the elevated of lipid peroxide levels in the ipsilateral 30 min after injection of iron solution by iron into the left cortex of rats. The fermented papaya preparation also increased superoxide dismutase activity in the cortex and hippocampus of them. These results suggest that the fermented papaya preparation has antioxidant actions and that it may be prophylactic food against the diseases associated with free radicals.
Yanbin Ji, Helmut Sies, and James A.Thomas
Deptartment of Biochemistry, Iowa State University, Ames, IA. and Institut für Physiologische Chemie I, Heinrich-Heine Universität, Dusseldorf, Germany
It has been suggested that the reaction of S-nitrosylated glutathione (GSNO) with proteins containing reactive sulfhydryls, leads to the formation of S-nitrosylated protein. It has also been shown that oxidation of protein sulfhydryls can produce S-glutathiolated proteins. In order to examine both of these protein modifications from a single reaction, the modified proteins forms generated in response to GSNO were separated by thin-gel electrofocusing under conditions that differentiate between protein forms with reduced sulfhydryls, with S-nitrosylated sulfhydryls, and with S-glutathiolated sulfhydryls. Glycogen phosphorylase b, a protein with two reactive sulfhydryls per subunit, was used as the model protein for these experiments.
Since S-nitrosylated protein isoforms have no additional charge on the modified sulfhydryl, these forms were separated after alkylating with iodoacetic acid, a negatively charged reagent. S-glu tathiolated protein isoforms have an additional single negative charge on the sulfhydryl and can be separated after alkylating with iodo acetamide, a neutral reagent. When phosphorylase b was incubated with freshly prepared GSNO the only modified forms of phosphorylase produced were S-nitrowever, after short periods of storage the GSNO caused formation of both S-nitrosylated and S-glu-tathiolated forms of phosphorylase. The longer GSNO was stored the more S-glutathiolated phosphorylase was formed. The S-glutathiola-tion could not be accounted for by glutathione disulfide present in the GSNO. HPLC analysis of the partially decomposed GSNO revealed the presence of substances with properties similar to those expected from the sulfenic acid of glutathione. It is proposed that thermal breakdown products of GSNO include a sulfenic acid form that is very effective as a protein S-glutathiolating agent.
T. Kaneyuki1, Y. Noda2, M. G. Traber2, A. Mori2, and L. Packer2
1Faculty of Health and Welfare Science, Department of Nutritional Science, Okayama Prefectural University, Okayama, Japan, 2Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3200, U.S.A.
Reactive oxygen species are associated with many degenerative diseases including cancer, cardio- and cerebrovascular diseases, immune system decline, brain dysfunction, and cataract formation. The antioxidant contents of fruits and vegetables may be a key factor in their beneficial effects. In this study, the water soluble components of fruits and vegetables was investigated for their free radical scavenging activity by ESR spin trapping methods. Lyophilyzed samples obtained from the extracts with distilled water (1:5, w/v) of 14 common vegetables were examined. Free radicals were generated by the Fenton reaction (.OH) and hypoxanthine-xanthine oxidase system (O2.), and were measured as DMPO-adduct. Eggplant, red, yellow and green bellpepper extracts showed potent superoxide anion scavenging activities (SOD-like activities). Ascorbate oxidase- or heat-treatment decreased the SOD-like activities in red bellpepper extract (67% or 62%), yellow bellpepper (67% or 64%) and green bellpepper (66% or 62%). SOD-like activity in eggplant extract did not decrease after ascorbate oxidase treatment, but decreased 40% by filtration with filters (<100,000 MW) and 65% (<10,000 MW). When eggplant was fractionated into epidermis and sarcoarp, epidermis extract exhibited the most potent hydroxyl radical scavenging and SOD-like activity, suggesting that SOD-like activity may be due to high molecular weight substances.
Hideaki Kabuto, Isao Yokoi, Nihei Yamamoto, Norio Ogawa, and Akitane Mori*
Department of Neuroscience, Institute of Molecular & Cellular Medicine, Okayama University School of Medicine, Okayama, Japan, and *Department of Molecular and Cell Biology, University of California, Berkeley, California, USA
Active oxygen species were generated and lipid peroxidation was induced in rat brain homogenates by incubation in the presence of iron ions, resulting in accumulation of thiobarbituric acid reactive substances (TBARS). In this study, we are examined the effects of magnetic fields(MF) on the generation of TBARS in the rat brain homogenate incubated with FeCl3.
Rats were anesthetized and perfused through the heart with ice-cold saline. Forebrains were removed and homogenized in 20 volumes of ice-cold Tris-HCl buffer (20 mM, pH 7.4). The homo genates were incubated with FeCl3 (50 microM) for 40 min at 37šC in various MFs, and the accumulated TBARS levels were compared with the control.
Sixty hertz of sine shaped MF (0.2-1.2mT) had no effect on the accumulation of TBARS. When the homogenates were incubated in static MF (2-4mT), the accumulation of TBARS was decreased. However, static MF at the magnitude of 1 and 5-300mT had no effect on the accumulation of TBARS.
These results indicate that the effectiveness of a magnetic field on iron ion induced active oxygen species generation in rat brain homogenates has a so called Œwindow¹ phenomenon, and that static magnetic fields might affect lipid peroxidation.
Tyrosine phosphorylation involvement in ADP mediated NF-kB activation
Nalini Kaul, Jinah Choi, and Henry Jay Forman
Department of Molecular Pharmacology & Toxicology, School of Pharmacy, University of Southern California, Los Angeles, CA-90033
ADP, a purinergic agonist is known to act through binding to P2Y receptors. Previous work from our laboratory has shown ADP stimulates the respiratory burst in alveolar macrophages. Also, we previously have shown NF-kB activation in alveolar macrophages and the J774A.1 mouse monocyte/macrophage cell line, upon treatment with 100 µM ADP for 30 min. Catalase abolished this ADP-stimulated NF kB activation, while SOD was ineffective (Free Rad Biol Med 21, 401 405,1996). Thus, H2O2 was the principal agent of the respiratory burst-associated NF-kB activation. The present study examined whether tyrosine phosphorylation was involved in the ADP mediated NF-kB activation by using the tyrosine kinase inhibitor genistein. The results suggest a complex involvement of tyrosine phosphorylation. Tyrosine phosphorylation was found to be inhibitory to basal NF-kB binding as genistein alone elevated NF-kB binding to a NF-kB probe. In contrast, ADP stimulated NF-kB activation was inhibited by genistein, suggesting tyrosine phosphorylation involvement in at least one other step opposing the effect of genistein on basal NF-kB binding. Interestingly, no clear effect of genistein was observed on PMA stimulated NF-kB activation. Tyrosine phosphorylation may be involved in the respiratory burst or in the events leading from the burst to NF-kB activation, consistent with the inhibition of the respiratory burst by genistein. Furthermore, genistein was also seen to inhibit the H2O2 stimulated NF-kB activation suggesting tyrosine phosphorylation involvement in the process downstream of the respiratory burst. Our results suggest tyrosine phosphorylation involvement in NF-kB activation at three steps: 1) ADP dependent respiratory burst in which tyrosine phosphorylation is a positive factor 2) H2O2 activation of NF-kB in which tyrosine phosphorylation is also a positive factor and 3) basal NF-kB binding, in which tyrosine phosphorylation is inhibitory.
Savita Khanna, Chandan K. Sen, Sashwati Roy, Marie-Odile Christen, and Lester Packer
Membrane Bioenergetics Group, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200 and Solvay Pharma, Suresnes Cedex 92151, France
Dithiolethiones occur naturally in several edible plant products, and have chemopreventive and detoxifying effects. Cabbage, brussel sprouts and other cruciferous vegetables have been found to contain significant amounts of dithiolethiones. Anethole dithiolethione (ADT) has been used clinically for many years as a choleretic and sialogogic agent without any adverse effects. More recently ADT has been shown to be a free radical scavenger and enhancer of intracellular GSH level. The protective effects of ADT against hydrogen peroxide or 4-hydroxynonenal (HNE)-induced cytotoxicity in human Jurkat T-cells were investigated. Jurkat T-cells were pretreated with ADT (10-50 mM) for 18 h and then challenged with glucose - glucose oxidase generated H2O2 or HNE for up to 4 h. Cytotoxicity was assessed by measuring i) leakage of lactate dehydrogenase from cells to medium, and ii) exclusion of the DNA intercalating fluorescent probe propidium iodide by viable cells. ADT content in cells and medium was measured following exposure of Jurkat T- cells to 10-50 mM ADT for 18 h. Higher concentrations of ADT exposure to cells resulted in higher accumulation of the compound in cells. The maximum level of ADT detected in cells was ~5 nmol/mg protein following 50 mM ADT exposure for 18 h.
Pretreatment of cells with ADT (10 or 25 M) for 18 h significantly protected cells against hydrogen peroxide or HNE-induced cytotoxicity. Treatment of cells with ADT (10-50 mM) for 72 h significantly increased the activities of catalase and glutathione reductase The maximum effect of ADT treatment on the activity of these enzymes was observed when cells were treated with 25 mM ADT for 72 h. Significant increases in GSH was observed in cells that were treated with ADT for 72 h. Using monobromobimane as a thiol probe we consistently observed that cells pretreated for 18 h with ADT (25 or 50 mM) also had an increased total thiol content. Exposure of Jurkat T- cells to H2O2 or HNE resulted in a time dependent decrease in cellular GSH. ADT (10-50 mM, 18 h) pretreatment circumvented H2O2 dependent lowering of cellular GSH. In conclusion, ADT is proving to be a potent cytoprotective thiol antioxidant with multifaceted mechanisms of action that suggest a remarkable therapeutic potential of the drug.