The effect of a synthetic neuromelanin on yield of free hydroxyl radicals generated in model systems

Neuromelanin is an amorphous pigment of the catecholamine origin that accumulates in certain dopaminergic neurons of the substantia nigra of human brain. In Parkinson's disease, there appears to be selective degeneration of the most heavily pigmented neurons of the substantia nigra, and this process has been linked to the presence of neuromelanin. It has been postulated that neuromelanin could increase the risk of oxidative stress reactions. On the other hand, melanin is usually considered to be an efficient antioxidant. Here we analyze experimental conditions that stimulate, or inhibit, antioxidant properties of neuromelanin. Using electron spin resonance (ESR)--spin trapping technique and salicylate hydroxylation assay, we monitored the formation of free hydroxyl radicals generated by a Fenton system in the presence of varying concentration of dopamine-melanin, a synthetic model for neuromelanin. Our data clearly indicate that the antioxidant action of neuromelanin is predominantly due to its ability to sequester redox-active metal ions such as iron. Using direct ESR spectroscopy, we have shown that ferric complexes with neuromelanin are resistant to reduction by mild biological reductants such as ascorbate. We have demonstrated that dopamine-melanin saturated with ferric ions, could enhance the formation of free hydroxyl radicals by redox activation of the ions. Thus, under the conditions that stimulate the release of accumulated metal ions, neuromelanin may actually become an efficient prooxidant. It is conceivable that neuromelanin, which normally is able to protect pigmented dopaminergic neurons against metal-ion related toxicity, could under extreme conditions have a cytotoxic role.     

Oxidative stress and the brain

Although the cause of Parkinson's disease is unknown, oxidative stress has been implicated in its pathogenesis. This theory postulates that normal metabolic processes in the nigrostriatal dopaminergic system may lead to loss of neurons, and that iron-dependent membrane lipid peroxidation may play an important role in the neuronal death. Recent research concerning iron-dependent lipid peroxidation is presented. First, catechols (including dopa and dopamine) and iron form strong oxidizing complexes and induce lipid peroxidation (LPO) in phospholipid liposomes. Active oxygen species including superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen, do not participate in this LPO, which is inhibited by an excess of dopa (dopamine). Cultured neurons and the substantia nigra are vulnerable to LPO. Second, synthetic melanin prepared by the autooxidation of catechols promotes LPO in the presence of iron. The effects of scavenging agents indicate that this LPO is mediated by superoxide, but not by other oxygen free radicals. Neuronal cell cultures are destroyed by this LPO. Third, catechols and superoxide produced by microglia cause the release of iron from ferritin. Microglia stimulated by phorbol myristate acetate produce superoxide and cause the release of iron from ferritin. Catechols also induce mobilization of ferritin iron. The released iron (i.e. loosely-bound iron) is available to iron-dependent LPO. These data suggest that the biochemical and morphological characteristics of the substantia nigra, which are concomitant with its functional role, provoke iron-dependent lipid peroxidation. It is essential to elucidate how iron bound loosely to low molecules comes into contact with catechols, neuromelanin and superoxide. Drugs that chelate iron site-specifically or modulate the microglial function may bring about some favorable changes in the disease process.     

Defective iron metabolism in genetic hemochromatosis. The mechanisms remain unknown in spite of genetic advances

Genetic haemochromatosis (GH) is one of the most common hereditary diseases, with a prevalence of 1-5/1000 in the Western world. In 90 per cent of cases a mutation is found in an MHC-class-like gene designated HFE, involving a substitution at position 282 of the HFE protein and resulting in defective binding of beta(2)-microglobulin. Animals with beta(2)-microglobulin deficiency develop iron overload, indicating this protein to be involved in the regulation of iron metabolism. Hepatic iron overload results in increased production of oxygen free radicals and peroxidation of membrane lipids, thus causing damage to lysosomes, mitochondria and the endoplasmic reticulum. These cellular events may progress to cell death, fibrogenesis, and the development of liver cirrhosis which is associated with a 200-fold increase in risk of hepatocellular carcinoma. In addition to the risk of diabetes, arthralgia, cardiac arrhythmia, pituitary insufficiency and hypogonadism, iron excess is also associated with aggravation of the cytotoxic effects exerted on hepatocytes by other agents such as alcohol or hepatotrophic viruses. The treatment of iron overload in GH consists of weekly venesection until the serum ferritin level is normalized, followed by maintenance therapy. Survival rates are normal if the disease is detected and treated before complications have developed.     

Iron as a hepatotoxin

Although essential for life, iron in excessive amounts may be toxic. The liver is particularly subject to the toxic effects of iron, since it is the major site of iron storage. Several inherited and acquired human disorders may result in hepatic iron overload, the most common of which are genetic hemochromatosis (GH) and transfusional iron overload. GH is an inherited disorder of iron metabolism, and in patients with GH excess iron absorbed from the gut is transported through the portal vein to the liver. The mechanisms by which excess iron exerts its cytotoxic effects include enhanced formation of free radicals and peroxidation of organelle membrane lipids. Lipid peroxidation can lead to structural and functional alterations in lysosomes, mitochondria and the endoplasmic reticulum. With massive iron overload, such iron-induced alterations may cause cell death, also known as sideronecrosis. At this stage, fibrogenesis is initiated and, if the excess iron is not removed, the increased deposition of collagen progresses to cirrhosis. However, the mechanisms underlying iron-induced fibrosis remain unclear. Transformation of fat-storing cells to collagen-producing myofibroblasts has been proposed to be induced either by iron; by lipid peroxides or other cellular factors released from iron-loaded, damaged hepatocytes; or by profibrogenic factors produced by activated Kupffer cells. In addition, iron may enhance the cytotoxic and, possibly, fibrogenic effects of other liver cell-damaging agents, such as alcohol or hepatotrophic viruses. Once cirrhosis is manifest, patients with GH demonstrate a 200-fold increase in the risk for development of hepatocellular carcinoma. In vitro, iron has been shown to possess mutagenic properties, but the results from in vivo models in which the genotoxic effects of iron overload have been studied are variable. Similarly, although iron has mitostimulatory effects on hepatocytes in vivo and preneoplastic cells in vitro, its role in tumor promotion and/or progression still remains unclear. Cirrhosis itself is of central importance in the carcinogenic process, but whether or not iron acts as an additional risk factor in this process, alone or by enhancing the tumorigenic properties of other hepatocarcinogens, has yet to be established.     

Infectious morbidity and defects of phagocytic function in end-stage renal disease: a review

Infectious diseases remain among the most morbid events and are an important cause of death in ESRD. These events are related to an acquired immunodeficiency that progresses during the development of uremic retention, as part of the broader spectrum, displayed by the "uremic syndrome". A central role in the hose defense against bacterial infection is played by the phagocytic polymorphonuclear white blood cells, which are characterized by the capacity to ingest bacteria (phagocytosis), which is followed by the destruction of those bacteria (killing capacity). This article reviews the mechanisms of development and the potential causes that have been held responsible for this aspect of the defective immune function. The observed changes are attributed to alterations in receptor expression, although more convincing evidence points in the direction of metabolic functional disturbances, especially in the NAD(P)H-oxidase-dependent production of oxygen free radicals. The most important causative factors are: uremic toxicity, iron overload, renal anemia, dialyzer bioincompatibility, and the type of renal replacement therapy. It is concluded that the phagocytic defect in ESRD is multifactorial and that each factor should be managed by specific therapeutic approaches.     

Prevention of mitochondrial injury by manganese superoxide dismutase reveals a primary mechanism for alkaline-induced cell death

Alkalosis is a clinical complication resulting from various pathological and physiological conditions. Although it is well established that reducing the cellular proton concentration is lethal, the mechanism leading to cell death is unknown. Mitochondrial respiration generates a proton gradient and superoxide radicals, suggesting a possible link between oxidative stress, mitochondrial integrity, and alkaline-induced cell death. Manganese superoxide dismutase removes superoxide radicals in mitochondria, and thus protects mitochondria from oxidative injury. Cells cultured under alkaline conditions were found to exhibit elevated levels of mitochondrial membrane potential, reactive oxygen species, and calcium which was accompanied by mitochondrial damage, DNA fragmentation, and cell death. Overexpression of manganese superoxide dismutase reduced the levels of intracellular reactive oxygen species and calcium, restored mitochondrial transmembrane potential, and prevented cell death. The results suggest that mitochondria are the primary target for alkaline-induced cell death and that free radical generation is an important and early event conveying cell death signals under alkaline conditions.     

Regulation of Ca2+ homeostasis by glucose metabolism in rat brain

Dopamine (DA) neurons are uniquely vulnerable to damage and disease. Their loss in humans is associated with diseases of the aged, most notably, Parkinson's Disease (PD). There is now a great deal of evidence to suggest that the destruction of DA neurons in PD involves the accumulation of harmful oxygen free radicals. Since the antioxidant hormone, melatonin, is one of the most potent endogenous scavengers of these toxic radicals, we tested its ability to rescue DA neurons from damage/death in several laboratory models associated with oxidative stress. In the first model, cells were grown in low density on serum-free media. Under these conditions, nearly all cells died, presumably due to the lack of essential growth factors. Treatment with 250 microM melatonin rescued nearly all dying cells (100% tau+ neurons), including tyrosine hydroxylase immunopositive DA neurons, for at least 7 days following growth factor deprivation. This effect was dose and time dependent and was mimicked by other antioxidants such as 2-iodomelatonin and vitamin E. Similarly, in the second model of oxidative stress, 250 microM melatonn produced a near total recovery from the usual 50% loss of DA neurons caused by neurotoxic injury from 2.5 microM 1-methyl-4-phenylpyridine (MPP+). These results indicate that melatonin possesses the remarkable ability to rescue DA neurons from cell death in several experimental paradigms associated with oxidative stress.     

Cytotoxicity of advanced glycation endproducts is mediated by oxidative stress

Non-enzymatic glycation of proteins with reducing sugars and subsequent transition metal catalysed oxidations leads to the formation of protein bound "advanced glycation endproducts" (AGEs). They accumulate on long-lived proteins and are for example structural components of the beta-amyloid plaques in Alzheimer's disease. Since the oxidation of glycated proteins as well as the interaction of AGEs with cell surface receptors produces superoxide radicals, it was tested in BHK 21 hamster fibroblast cells and SH-SY5Y human neuroblastoma cells if AGEs can exert cytotoxic effects on cells. Cell viability was assessed with three independent tests: MTT-assay (activity of the mitochondrial respiratory chain), lactate dehydrogenase assay (release of cytoplasmatic enzymes, membrane integrity) and Neutral Red assay (active uptake of a hydrophilic dye). Two model AGEs, chicken egg albumin-AGE and BSA-AGE, both caused significant cell death in a dose-dependent manner. The cytotoxic effects of AGEs could be attenuated by alpha-ketoglutarate and pyruvate, by antioxidants such as thioctic acid and N-acetylcysteine, and by aminoguanidine, an inhibitor of nitric oxide synthase. This suggests that reactive oxygen species as well as reactive nitrogen species contribute to AGE mediated cytotoxicity. Since AGEs accumulate on beta-amyloid plaques in AD over time, they may additionally contribute to oxidative stress, cell damage, functional loss and even neuronal cell death in the Alzheimer's disease brain.     

A scientific rationale for protective therapy in Parkinson's disease

The desire to introduce neuroprotective therapy for Parkinson's disease has begun to focus attention on pathogenetic mechanisms responsible for cell death. Considerable theory and some evidence have now accumulated to suggest that factors related to oxidative stress, mitochondrial bioenergetic defects, excitatory neurotoxicity, calcium cytotoxicity, and trophic factor deficiencies acting either singularly or in combination may contribute to the development of cell death in Parkinson's disease. A better understanding of the specific pathogenetic mechanism involved in cell degeneration might provide a scientific basis for testing a putative neuroprotective therapy. This chapter reviews the theory and evidence in support of these different mechanisms and possible strategies that might provide neuroprotection and interfere with the natural progression of Parkinson's disease.     

Free radicals and antioxidants

The data obtained from the author's laboratory were used to make this review. The author's classification of free radicals, approaches, the origin and metabolism of primary radicals, the contribution of iron ions to the production of secondary radicals and the mechanisms of antioxidative protection of cells and tissues from damage are considered. According to the classification proposed, the radicals may be divided into primary (superoxide, semiquinones and nitric oxide), secondary (hydroxyl and lipid radicals) and tertiary (radicals of antioxidants). The primary radicals are formed by enzymatic systems and perform biologically important functions. The secondary radicals are formed from hydroperoxides in the reactions of divalent iron ions and damage to cell structures. In the cells and blood plasma, there is a complicated system of antioxidants that prevent the production of secondary radicals. All antioxidants may be arbitrarily divided into water-soluble and hydrophobic. The first group involves the enzymes catalase and glutathione peroxidase, iron ion chelators (such as ceruloplasmin and transferrin in the blood and carnosine in other tissues), and, probably, hydroxyl radical traps, such as uric acid and ascorbate. The hydrophobic antioxidants include primarily the free radical traps alpha-tocopherol, flavonoids, and carotenes. Studies of lipid peroxidation kinetics in the membranous structures, carried out by chemiluminescence and mathematical modeling of the reactions have shown that the radicals of antioxidants (such as alpha-tocopherol) enter the further reactions in the lipid phase, including those with lipid hydroperoxides.     

Free radicals, oxidative stress and antioxidant vitamins

Free radicals having oxidizing properties are produced in vivo. The monoelectronic reduction of dioxygen generates the superoxide radical (.O2-) which, according to the experimental conditions, behaves as a reducing or an oxidizing agent. Its dismutation catalyzed by superoxide dismutases (SODs) produces hydrogen peroxide. The latter reacting with .O2- in the presence of "redox-active" iron produces highly aggressive prooxidant radicals, such as the hydroxyl radical (.OH). This production is prevented through intracellular enzymes (catalase and glutathione peroxidases) which destroy the hydrogen peroxide involved in the biosynthesis of .OH. An increase in SODs activity without parallel enhancement of the enzymes destroying H2O2 may lead to important cellular disturbances. Other enzymes acting with glutathione as substrate (especially glutathione S-transferases) contribute to the antioxidant defence. The same holds true for selenium and zinc which act mainly through their involvement in the structure of both antioxidant enzymes and nonenzymatic proteins. Another line of antioxidant defence is represented by substrates acting as chain-breaking antioxidants in destructive processes linked to prooxidant free radicals, such as lipid peroxidation. The main membranous antioxidant is alpha-tocopherol which is able to quench efficiently lipid peroxyl radicals. Its efficiency would be quickly exhausted if the tocopheryl radical formed during this reaction wouldn't be retransformed into alpha-tocopherol through the intervention of ascorbate and/or glutathione. Ubiquinol and dihydrolipoate also contribute to the membranous antioxidant defence, whereas carotenoids are mainly responsible for the prevention of the deleterious effects of singlet oxygen. An oxidative stress is apparent when the antioxidant defence is insufficient to cope with the prooxidant production.     

Imbalance of oxygen activation and energy metabolism as a consequence or mediator of aging

Ever increasing numbers of aging theories suggest that free radicals are only one factor among others that may initiate stochastic disorders finally terminating life. It is therefore compelling not only to demonstrate the existence of increasing steady-state concentrations of free oxygen radicals during senescence, but it is essential to show that they act in concert with other postulated triggering factors of aging. We have recently shown that various factors may have a life-long influence and challenge oxygen homeostasis of cell respiration. Among these factors are environmental pollutants, therapeutics, and transient hypoxia. Although the nature of these "hits" is different, mitochondrial respiration was found to respond in a similar manner to each of them. The major derangement was an univalent electron leak to oxygen giving rise to the establishment of oxidative stress. Associated with this transformation, oxidative phosphorylation was impaired with the resultant reduction of cellular ATP. Mitochondria from senescent rats exhibited similar alterations of all cell parameters found when adult animals were exposed to "environmental stress" or transient ischemia. Age-related stimulation of mitochondrial oxygen radical generation is therefore suggested to result from accumulation of minihits during life. Based on our data, together with those from other laboratories, it is possible to assess the ranking order of oxygen radicals in the development of stochastic events associated with (or causing) aging.     

Spinal anesthesia with bupivacaine and fentanyl in geriatric patients

In Escherichia coli, fur mutants that constitutively express their native iron chelating agent, enterobactin, are significantly more sensitive to near-UV radiation (NUV) than wild type, An entA mutant, which is incapable of synthesizing enterobactin, is equal to wild type in resistance to NUV irradiation. However, the addition of Fe+3 enterobactin but not AI+3 enterobactin to entA cell suspensions just prior to irradiation results in an increased sensitivity to NUV irradiation. A fes mutant, which is unable to reduce and release iron from enterobactin, is significantly more sensitive to NUV irradiation than wild type. The addition of nontoxic levels of H2O2 (5 microM) just prior to irradiation significantly increases sensitivity of both fur and fes mutants. These results suggest that one mechanism by which NUV irradiation leads to cell lethality is by creating a transient iron overload, producing very favorable conditions for the production of highly deleterious free radicals through a variety of mechanisms that lead to oxidative stress and DNA damage including lethal and mutagenic lesions. These results are consistent with the hypothesis that enterobactin is an endogenous chromophore for NUV and contributes to cell lethality via the destruction of its ligand, releasing Fe+2 into the cytoplasm to catalyze the production of highly reactive hydroxyl radicals and other toxic oxygen species via the Haber-Weiss reaction.     

Social anxiety in children with anxiety disorders: relation with social and emotional functioning

BACKGROUND: Oxygen radicals have been implicated as important mediators in the early pathogenesis of acute pancreatitis, but the mechanism by which they produce pancreatic tissue injury remains unclear. We have, therefore, investigated the effects of oxygen radicals on isolated rat pancreatic acinar cells as to the ultrastructure, cytosolic Ca2+ concentration and energy metabolism. METHODS: Acinar cells were exposed to an oxygen radical-generating system consisting of xanthine oxidase, hypoxanthine and chelated iron ions. Cell injury was assessed by LDH release and electron microscopy. Cytosolic Ca2+ levels and mitochondrial membrane potential were determined by flow cytometry; adenine nucleotide concentrations by HPLC. Mitochondrial dehydrogenase activity was measured by spectrophotometric assay. RESULTS: Oxygen radicals damaged the plasma membrane as shown by a 6-fold LDH increase in the incubation medium within 180 min. At the ultrastructural level, mitochondria were the most susceptible to oxidative stress. In correlation to the pronounced mitochondrial damage, the mitochondrial dehydrogenase activity declined by 70%, whereas the mitochondrial membrane potential was enhanced by 27% after 120 min. Together this may cause the 85% decrease in the ATP concentration and the corresponding increase in ADP/AMP observed in parallel. In addition, an immediate 26% increase in cytosolic Ca2+ was found, a change which could be inhibited by BAPTA, reducing cellular damage. CONCLUSION: Cytosolic Ca2+ synergizes with oxygen radicals causing alterations of the ultrastructure and energy metabolism of acinar cells which might contribute to the cellular changes found in early stages of acute pancreatitis.     

In vivo effects of fumonisin B1-producing and fumonisin B1-nonproducing Fusarium moniliforme isolates are similar: fumonisins B2 and B3 cause hepato- and nephrotoxicity in rats

Orellanine, [2,2'-bipyridine]-3,3',4,4'-tetrol-1,1'-dioxide, is the toxin responsible for the lethal nephrotoxicity of some Cortinarius mushrooms. Our present ESR and spin-trapping studies of the redox properties of the system of non-illuminated orellanine, ferrous iron and dioxygen contribute to understanding the molecular mechanism of its toxicity. UV-visible spectrophotometry, cyclic voltammetry and ESR in frozen medium showed the formation of a wine-red tris complex, Fe(III)Or3. This ferric complex is easily reducible (Ep = -565 mV vs Ag/AgCl/3M KCl at pH 7), involving a one-electron reversible process. Spin-trapping using DMPO is employed to detect the generation of superoxide anion and hydroxyl radicals. The instantaneous one-electron oxidation of ferrous ions in the presence of the toxin under air is concomitant with dioxygen consumption as supported by dioxygen consumption. GSH involves the toxin and ferrous ions under air in a redox cycling process resulting in the production of glutathionyl and oxygen free radicals, observed for the first time with an iron complex of a mushroom toxin. In most cases, EDTA is not able to prevent the Fe(III)Or3 and radical formation. The ortho-dihydroxylated groups borne by the di-N-oxidized bipyridine structure and not the bipyridine structure itself, are responsible for the formation of a stable ferric complex at pH 7, as they are for the generation of an apparently stable ortho-semiquinone anion radical. These one-electron mechanisms may play a major role in some of the known toxic effects of orellanine.     

Toxicologic importance of iron and copper atoms and their relation to reactive oxygen metabolites

Free radicals are, on the one hand, necessary for the physiological function of some systems but, on the other had, they play an important pathologic role. The formation of free radicals can be the result of various conditions and can initiate various diseases. Their formation may also be a consequence of certain pathological state of the organism. In this way generated free radicals can cause in the secondary to the damage organism. The metabolism of free radicals is significantly influenced by transition metals. These metals are present in the organism at given oxidative state chelated in the proteins. In this form the metalloproteins to have unique catalytical and redox properties. The transition metals are a part of an active centre of many enzymes. Iron and copper are the predominant transition metals in human organism. These metals are vitally important, but if present in high concentration, in unsuitable oxidative state and at improper site, they can catalyse the formation of highly toxic reactive metabolities of oxygen, for example hydroxyl radicals. The toxic damage may be direct if the metals are present in high oxidative state Fe(IV) or Fe(V). These "ferryl" compounds are strong prooxidants. The organism maintains the iron metabolism in equilibrium. If the iron plasma concentration reaches 40 micro-mol/L, it is a sign, that iron is released from physiological protein structures and forms so called "catalytically active iron". In this form iron can be involved in Fenton reaction in which hydroxyl radical is formed. The article discussed the toxic effect of "catalytically" active iron at a given oxidative state with its influence on some diseases.     

Melatonin prevents death of neuroblastoma cells exposed to the Alzheimer amyloid peptide

Studies from several laboratories have generated evidence suggesting that oxidative stress is involved in the pathogenesis of Alzheimer's disease (AD). The finding that the amyloid beta protein (Abeta) has neurotoxic properties and that such effects are, in part, mediated by free radicals has provided insights into mechanisms of cell death in AD and an avenue to explore new therapeutic approaches. In this study we demonstrate that melatonin, a pineal hormone with recently established antioxidant properties, is remarkably effective in preventing death of cultured neuroblastoma cells as well as oxidative damage and intracellular Ca2+ increases induced by a cytotoxic fragment of Abeta. The effects of melatonin were extremely reproducible and corroborated by multiple quantitative methods, including cell viability studies by confocal laser microscopy, electron microscopy, and measurements of intracellular calcium levels. The importance of this finding is that, in contrast to conventional antioxidants, melatonin has a proposed physiological role in the aging process. Secretion levels of this hormone are decreased in aging and more severely reduced in AD. The reported phenomenon may be of therapeutic relevance in AD.     

Treatment of postprostatectomy urinary incontinence with behavioral methods

Neurodegeneration is characterized by a marked accumulation of iron in the affected brain regions. The reason for this is still unknown. In this article we review the available data on the possible involvement of iron and mediated oxidative stress in the aetiology of Parkinson's disease and related disorders. Iron chelators, if they effectively prevent radical formation, have great therapeutic potential against ischaemia/reperfusion, rheumatoid arthritis, and anthracycline toxicity, which are most likely free radical-mediated. The efficacy of the best established chelating drug desferal in neurodegenerative disease is limited due to its high cerebro- and oculotoxicity. New bioactive chelating agents are currently being developed, among them are oxidative stress activatable iron chelators which are most likely less toxic and can flexibly respond to an increase of free radical formation in the cell.