Effect of haloperidol on brain mitochondrial respiration and nitric oxide and superoxide production
Silvia Lores Arnaiz, María Florencia Coronel, and Alberto Boveris
Laboratory of Free Radical Biology. School of Pharmacy and Biochemistry, University of Buenos Aires, Jun¹n 956, 1113 Buenos Aires, Argentina
Haloperidol, a widely used neuroleptic drug, causes a variety of neurological side effects including various movement disorders. It has been suggested that inhibition of mitochondrial respiration and free radical induction may be involved in haloperidol neurotoxicity. In this study, mice were injected i.p. according to two different treatments: a) a single injection (1 mg/kg), sacrificed 1 hour after the injection (single-dose model), and b) two injections (1 mg/kg each) at 10 a.m. and at 4 p.m sacrificed 24 hours after the first dose (double-dose model). Determinations of oxygen consumption were carried out in isolated mitochondria, in the presence of either malate-glutamate or succinate (state 4) and the same substrates plus ADP (state 3). Superoxide and nitric oxide production were measured in submitochondrial particles (SMP).
Single-dose haloperidol treatment produced a 33% inhibition in malate-glutamate-dependent respiration rate (state 4), one hour after drug injection. No significant changes were found in malate-gluta mate-dependent oxygen consumption after 24 hours (double-dose treatment). No changes were observed in succinate-supported respiration rates of mitochondria from treated mice, as compared with controls. Superoxide production in the presence of NADH and rotenone was increased by 248% in brain SMP from single-dose treated mice (control value: 5.0 ± 0.5 nmol/min.mg protein). Nitric oxide production was inhibited by 39 % and 54% in SMP from haloperidol-treated mice (single- and double-dose treatments respectively) (control value: 1.6 0.2 nmol/min.mg protein).
Our results suggest that haloperidol neurotoxicity would be mediated by inhibition of mitochondrial electron transfer and enhancement of superoxide production at the NADH dehydrogenase site. This inhibition does not seem to be caused by increased ONOO formation.
G. Lu, E.L. Greene, T. Nagai,, and B.M. Egan
Departments of Medicine, Clinical Pharmacology, and Pharmacology Medical University of South Carolina, Charleston, South Carolina
Obese hypertensive patients with cardiovascular risk factor clustering and increased risk for atherosclerotic disease have increased plasma non-esterified fatty acid (NEFA) levels. Oleic acid is a prototypical NEFA and is the most abundant NEFA in human plasma. Our previous studies demonstrated that oleic acid induced a mitogenic response in rat aortic smooth muscle cells (RASMC) through PKC and ERK-dependent pathways (Lu and Egan). In the current set of studies we tested the hypothesis that oleic acid activates mitogenesis through an oxidant sensitive signal transduction pathway. We investigated the effect of oleic acid on activation of mitogen-activated protein kinases ERK1 & ERK2, and the generation of reactive oxygen species (R.O.S.) by using the oxidant sensitive fluorescent probe 2¹,7¹- dichlorofluorescin diacetate (DCFH-DA). Relative fluorescence intensity and fluorescent images were obtained over time (1-5 minutes) using LASER confocal scanning microscopy. 100 µM oleic acid induced a time-dependent increase of H2O2 8-fold when compared to control cells. The peak signal was observed by 5 minutes. Oleic acid induced increases in R.O.S. were blocked by the PKC inhibitor bisidolylmailemide and PKC downregulation with phorbol 12-acetate, 13-myristate (PMA). Stearic and elaidic acids, which are weak PKC activators, did not significantly increase H2O2 production. The increases of H2O2 in response to oleic acid were inhibited by N-acetyl-cysteine (NAC). NAC also inhibited ERK activation and cell proliferation by oleic acid. These data suggests that the generation of R.O.S. in RASMCs exposed to oleic acid is a PKC-dependent event. Reactive oxygen species emerge as a potential critical signaling event between the activation of PKC and ERK in VSMC stimulated with oleic acid. In addition, these observations raise the possibility that the elevated plasma non-esterified fatty acids, oleic acid in particular, may contribute to vascular growth and remodeling in atherosclerosis by a PKC-dependent mechanism to generate reactive oxygen species which subsequently activate the ERKs.
Oxidative status of rat sperm cells exposed to iron
Florencia Lucesoli, Marina Caligiuri, and Cesar G. Fraga
Physical Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina
Oxidative damage to sperm cells could be important in the etiology of male infertility, birth defects, and inheritable mutations. In this study, we characterized the susceptibility of rat sperm cells to an oxidative insulting an in vitro system in which oxidative stress was generated by incubating sperm cells with ferrous sulfate (50 µM) for different periods of time (0-60 min). Sperm motility and plasma membrane integrity (measured by an hypo osmotic test) decreased after 30 min of incubation, however the cells remained viable for 60 min. When compared to controls, the following changes were observed in indicators of oxidative damage: a) an increase in iron accumulation in sperm cells (3.5-folds); b) a modest increase in iron-dependent accumulation of TBARS within the cells (22%); c) an increase in TBARS release into the incubation medium (4.9-fold); d) a decrease in cells alpha-tocopherol (70%), ubiquinol-9 (7%); and ubiquinol-10 (36%); e) an increase in cells protein-associated carbonyls (55%); and f) a decrease in cells protein sulphydryl groups (25%). By analyzing the kinetics of oxidation products formation and antioxidants consumption, it is possible to establish a sequential order for the oxidation of cellular components of rat sperm cells that occurs under oxidant conditions.
Supported by grants UBA (FA030), and CONICET (PIA 6114/96) to CGF
Jens Lykkesfeldt, Tory M. Hagen, Vladimir Vinarsky and Bruce N. Ames
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
Cellular response to increased oxidative stress was investigated in rat hepatocytes. Recycling and biosynthesis of ascorbic acid from dehydroascorbic acid and gulono-1,4-lactone, respectively, were used as measures of response capacity to an elevated oxidant load induced by tert-butylhydroperoxide. Hepatocytes isolated freshly from old rats exhibited a significant inverse correlation between oxidant load and ascorbic acid recycling capacity (P < 0.005). Ascorbic acid biosynthesis in these cells was unaffected by various concentrations of tert-butylhydroperoxide but amounted to only about half of the biosynthetic rate when compared to cells from young animals (P < 0.001). Additionally, the hepatic ascorbic acid concentration was 54 % lower in cells from old rats when compared to cells isolated from young animals (P < 0.0005). Cells from young animals were not significantly affected by the tert-butylhydroperoxide treatments. The results demonstrates a declining ability with age to respond to increased oxidative stress.
(R)-a-Lipoic acid is a powerful antioxidant and the preferred stereoisomer of the mitochondria. A two-week dietary supplementation of old animals with 0.5 % (R)-a-lipoic acid prior to cell isolation almost completely reversed the age-associated effects on ascorbic acid availability and metabolism. These results provide further evidence for the involvement of mitochondrial deterioration in aging and its role in cellular response to environmental stresses. However, more work is required to understand and further characterize the cellular mechanisms that cause this astounding effect as well as the role of mitochondria in aging.
S-Glutathiolation of H-Ras in NIH 3T3 cells
Robert J. Mallis and James A. Thomas
Department of Biochemistry and Biophysics, Iowa State University, Ames, IA
The signal transduction protein H-Ras is a critical link in activation of the MAP kinase signaling cascade in both normal and oncogenic cells. Additionally, H-Ras has been implicated both in activation of superoxide producing factors and in transducing oxidative signals through the MAP kinase pathway. Because S-glutathiolation of proteins is one of the first reactions to occur in cells after oxidative insult, it is hypothesized that one of the functions of the H-Ras protein is to respond to an oxidation event through S-glutathiolation of one of its three surface-exposed cysteine residues.
Purified H-Ras was shown to be readily S-thiolated in vitro when reacted with glutathione disulfide. It was also found to react with diamide or hydrogen peroxide in the presence of reduced glutathione. When reduced glutathione was not present in the latter two reactions, formation of intersubunit dimers was seen. While it is important to establish reactivity of a protein in vitro, it is essential to determine whether and to what extent a protein is modified in the context of a cellular system. NIH 3T3 cells stably transfected with two different forms of the H-Ras protein, the wildtype and the C118S mutant, were labeled with 35S and H-Ras was immunoprecipitated from cells which had been exposed to 2mM diamide for 2 minutes. In this context, DTT-reducible counts were found to comigrate with the immuno precipitated H-Ras, showing S-glutathiolation of both wildtype H-Ras and the C118S mutant in a cellular context in response to an oxidative stress. These data suggest that H-ras cysteines normally involved in S-palmityolation may become S-glutathiolated during oxidative stress. In addition to diamide, the S-glutathiolation potential of Cys-NO, AAPH and hydrogen peroxide were tested in this system.
Lucia Marcocci1,2, Laura McLaughlin1, Michael Kobayashi1, and Lester Packer1
1Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. 2Department of Biochemical Sciences ³A. Rossi Fanelli² and CNR Center of Molecular Biology, University of Rome ³La Sapienza² Rome, Italy
Cellular reduction of a-lipoic acid (LA) to dihydrolipoic acid (DHLA) is important for its antioxidant properties. Several pathways of DHLA formation from LA have been documented: e.g. the NADH-dependent reduction by lipoamide dehydrogenase (LAD) specific for R-LA, or the NADPH-dependent reduction by glutathione reductase (GR) or by purified thioredoxin reductase (TR). Catalytic activity of TR in cells has been reported to be regulated by selenium availability. Thus in order to explore possible strategies for the modulation of LA reduction, the effects of selenium supplementation on the cellular formation of DHLA from LA was investigated. A human T- lymphocyte cell line (Jurkat) was grown for at least two weeks in RPMI 1640 medium containing 5% low selenium fetal bovine serum, L-glutamine, pyruvate, antibiotics, and with or without 40 nM added sodium selenite (Se). Intact cells or cell homogenates were then incubated for up to 2 hrs at 37°C with 2 mM R, S, or racemic LA in PBS containing 5mM glucose, or 1mM of either NADH or NADPH. At various intervals of time, DHLA formation was then measured by HPLC using electrochemical detection (ECD). Se-supplemented samples had, with respect to Se-deficient controls, a 3 fold increase in the catalytic activity of TR and a 5 fold increase in the activity of glutathione peroxidase (GSH-Px); however, the activities of GR and LAD were not affected by Se-supplementation. The glucose-dependent rate of reduction of R, S or racemic LA was 40% faster in Se-supple mented than in Se-deficient intact cells. In cell homogenates from Se-supplemented cells, the reduction rate of R, S, or racemic LA in the presence of 1 mM NADPH was 70% higher than in homogenates from Se-deficient cells. On the other hand, in the presence of 1 mM NADH, the reduction of LA in cell homogenates was not affected by Se-supplementation and was specific for R-LA. Our studies clearly indicate that supplementation with sodium selenite improves the ability of Jurkat cells to reduce either R or S-LA to DHLA by a NADPH-dependent pathway. Taking into consideration that although the activity of some other selenium dependent enzyme cannot be excluded, coupled with the fact that under our experimental conditions LA was not reduced by GSH-Px, a key role for TR in cellular reduction of LA is likely.
*F. Marotta, P. Safra, H. Tajiri, and ÝI. Reizakovic
GI service, S. Anna Hospital, Como, Italy; * Econum Lab., Villeneuve d¹Ascq, France; / Natl. Cancer Center East, Chiba, Japan; ÝLiver Unit, Niguarda Hospital, Milano, Italy
The aim of this investigation was to study the oxidative phenomena taking place in the early recovery phase after alcohol withdrawal. Further, the effects of a novel natural antioxidant, i.e. Bionormalizer (BN), in such clinical setting was studied. Forty-six alcoholics (HBV and HCV negative) with moderate drinking habits (daily ethanol intake: >80g - <120g) were enrolled and divided in two groups given either placebo or 9g/nocte of BN by mouth for one week. Patients agreed to stop alcohol intake and daily blood sampling was obtained for routine tests and to check plasma and erythrocyte level of MDA, SOD, GPX and hydroperoxide level. Groups were comparable as for initial routine blood biochemical and antioxidant parameters (selenium, a-tocopherol, ascorbic acid and erythrocyte GPX), as well as smoking habit. BN Two patients on BN were later excluded for protocol violation. BN prevented the early increase of plasma TBARS observed in placebo group enabling a near-to-normal level also of erythrocyte MDA already on the fourth day. BN also prevented the significant drop of erythrocyte GPX and the transient decrease of plasma SOD observed in placebo group. Despite alcohol withdrawal, plasma lipid hydroperoxide remained significantly elevated in placebo group but this phenomenon was rapidly improved by BN. These data suggest that a pro-oxidative condition with an avid consumption of SOD and glutathione still takes place once alcohol ingestion is stopped while confirming the enhanced susceptibility to oxidative stress due to derangement of structural membrane lipids in patients with alcohol-related liver disease. BN is able to significantly prevent free radical-mediated lipoperoxidative changes occuring soon after alcohol withdrawal while fastening the recovery mechanisms. Alcoholics would potentially benefit from increased dietary supplementation of truly effective natural free radicals-scavengers, such as Bionorma lizer.
Free radical production during the reaction of cytochrome P450 with linoleic acid hydroperoxide
Ronald P. MasonÝ, David P. BarrÝ, Aldo Tomasi*, and Cristina Rota*
*Department of Biomedical Sciences, University of Modena, via Campi 287, 41100 Modena, Italy and ÝNational Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, 27709, USA
The ESR spin-trapping technique was employed to investigate the reaction of rabbit cytochrome P450 (P450) 1A2 with linoleic acid hydroperoxide (13-HPODE). This system was compared with chemical systems where ferrous sulfate or ferric chloride was used in place of P450. The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 2-methyl-2-nitrosopropane (MNP) were employed to detect and identify radical species.
Reacting P450 with 13-HPODE in presence of MNP, carbon centered radical adducts were detected. To better characterize them, linoleic acid hydroperoxide synthesized from deuterated linoleic acid (9,10,12,13-d4 linoleic acid) was also employed. Under aerobic conditions we detected two epoxy allilic radicals, while under anaerobic conditions we detected a third radical species that we could not assign.
Hydroxyl, superoxide, peroxyl, methyl, and acyl radicals were detected in the P450 system using DMPO as spin trap. The same DMPO radical adducts were detected in the FeSO4 system. Only DMPO/OH radical adduct and carbon-centered radical adducts were detected in the FeCl3 system. peroxyl radical production was completely O2-dependent.
Based on our results, we propose that polyunsaturated fatty acids are initially reduced to form alkoxyl radicals, which then undergo intramolecular rearrangement to form epoxyalkyl radicals. Each epoxyalkyl radical reacts with O2, forming a peroxyl radical. Subsequent unimolecular decomposition of this peroxyl radical eliminates superoxide anion radical.
Ronald P. Mason*, Herbert J. Sipe§, Jr., David P. BarrÝ, Jose G. Martinez*, and Bradley E. Sturgeon*
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Sciences, National Institutes of Health, Research Triangle Park, NC 27709, §Hampden-Sydney College, Department of Chemistry, Hampden-Sydney, VA 23943, ÝBruker Instruments, 19 Fortune Drive, Manning Park, Billerica, MA 01821
The anitoxidant properties of ascorbate have been known for quite sometime. Cellular systems contain as much as a millimolar concentration of both ascorbate and glutathione (GSH). It has been proposed that GSH and superoxide dismutase (SOD) act in a concerted effort to eliminate biologically generated radicals. The tyrosine phenoxyl radical (tyr.) generated by a peroxidase system (HRP/H2O2/tyrosine) will react with GSH to form the glutathione thiyl radical (GS.) which can be detected by spin trapping with 5,5-dimethyl pyroline N-oxide (DPMO) to form DPMO/.SG. Under physiological conditions, the GS. will react with the glutathione anion (GS) to form the disulfide radical anion ([GSSG.]). This highly reactive disulfide radical anion will reduce molecular oxygen forming superoxide. In a concerted effort SOD will dismutate superoxide resulting in the elimination of the radical. The GSH chemistry involved is well documented and accounts for the consumption of oxygen by thiol radicals. Nevertheless, the physiological relevance of this GSH/SOD concerted effort is questionable. In a tyrosine phenoxyl radical-generating system containing ascorbate as well as GSH, the ascorbate completely eliminates oxygen consumption and diminishes GS. formation and when measuring the ascorbate radical directly using fast-flow ESR only minor changes in the ascorbate radical ESR signal intensity occur in the presence of GSH. These result indicate that in the presence of ascorbate, GSH-generated superoxide is not a significant species in the detoxification pathway of biologically generated radicals.
Ronald P. Mason, Richard A. Tshcirret-Guth, H. Ewa Witkowska, Yang C. Fann, David P. Barr, Paul Ortiz de Montellano, and Michael R. Gunther
Laboratory of Pharmacology and Chemistry, National Insitute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, Department of Pharmaceutical Chemistry, University of Southern California, San Francisco, CA 94143-0446, and Children¹s Hospital Oakland Research Institute, Oakland, CA
The reaction between metmyoglobin and hydrogen peroxide produces both a ferryl-oxo heme and a globin-centered radical(s) from the two oxidizing equivalents of the hydrogen peroxide. Evidence has been presented for localization of the globin-centered radical on one tryptophan residue and tyrosines 103 and 151. When the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is included in the reaction mixture, a radical adduct has been detected, but the residue at which that adduct is formed has not been determined. Replacement of either tryptophans 7 and 14 or tyrosines 146 and 151 with phenylalanine has no effect on the formation of DMPO adduct in the reaction with hydrogen peroxide. When tyrosine 103 is replaced with phenylalanine, however, only DMPOX, a product of the oxidation of the spin-trap, is detected. Tyrosine-103 is, therefore, the site of radical adduct formation with DMPO. The spin trap 2-methyl- 2-nitrosopropane (MNP), however, forms radical adducts with any recombinant sperm whale metmyoglobin that contains either tyrosine 103 or 151. Detailed spectral analysis of the DMPO and MNP radical adducts of isotopically substituted tyrosine radical yield complete structural determinations. The multiple sites of trapping support a model in which the unpaired electron density is spread over a number of residues in the population of metmyoglobin molecules, at least some of which are in equilibrium with each other.