Oxyradical Production Research Articles

Contributions of mitochondrial alterations, resulting from bad genes and a hostile environment, to the pathogenesis of Alzheimer's disease

This chapter discusses different genetic and environmental factors that either cause, or increase the risk of, Alzheimer's Disease (AD). Each genetic causal or predisposition factor promotes increased oxidative stress and disrupts cellular calcium homeostasis in neurons. Mitochondrial function is compromised by oxyradicals and calcium overload resulting in further oxyradical production, impaired calcium homeostasis, and activation of apoptotic caspases. Age-related DNA damage (nuclear and mitochondrial) may endanger neurons and render them vulnerable to excitotoxicity and apoptosis. Environmental factors that may predispose to AD include excessive caloric intake, folic-acid insufficiency, and disuse of neural circuits involved in learning and memory. Novel preventative approaches for AD based upon the mechanisms include dietary restriction, mental aerobics, and dietary supplementation with folic acid and certain anti-oxidants . Therapeutic strategies may include antioxidants, anti-inflammatory agents, and drugs that stabilize mitochondrial function.

Energetics and oxidative stress in synaptic plasticity and neurodegenerative disorders.

As in other cells, neurons use adenosine triphosphate (ATP) as an energy source to drive biochemical processes involved in various cell functions, and produce reactive oxygen species (ROS) as "by products" of oxidative phosphorylation. However, the electrical excitability and structural and synaptic complexity of neurons present unusual demands upon cellular systems that produce or respond to ATP and ROS. Mitochondria in axons and presynaptic terminals provide sources of ATP to drive the ion pumps that are concentrated in these structures to rapidly restore ion gradients following depolarization and neurotransmitter release. Mitochondria may also play important roles in the regulation of synaptic function because of their ability to regulate calcium levels and ROS production. ROS generated in response to synaptic activity are now known to contribute to the regulation of long-term structural and functional changes in neurons, and the best-known example is the nitric oxide radical. The high-energy demands of synapses, together with their high levels of ROS production, place them at risk during conditions of increased stress, which occur in aging, neurodegenerative disorders such as Alzheimer's and Parkinson's diseases, and after acute traumatic and ischemic insults. Energy depletion and/or increased oxidative damage to various synaptic proteins can result in a local dysregulation of calcium homeostasis and synaptic degeneration. Accordingly, recent studies have shown that dietary and pharmacological manipulations that improve energy efficiency and reduce oxyradical production can prevent synaptic degeneration and neuronal death in experimental models of neurodegenerative disorders. A better understanding of the molecular control of subcellular energy production and utilization, and of the functional relationships between energy metabolism, ion homeostasis, and cytoskeletal and vesicular dynamics, will provide novel insight into mechanisms of neuronal plasticity and disease.

Iodoacetate protects hippocampal neurons against excitotoxic and oxidative injury: involvement of heat-shock proteins and Bcl-2.

Mild metabolic stress may increase resistance of neurons in the brain to subsequent, more severe insults, as demonstrated by the ability of ischemic pre-conditioning and dietary restriction to protect neurons in experimental models of stroke- and age-related neurodegenerative disorders. In the present study we employed iodoacetic acid (IAA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, to test the hypothesis that inhibition of glycolysis can protect neurons. Pre-treatment of cultured hippocampal neurons with IAA can protect them against cell death induced by glutamate, iron and trophic factor withdrawal. Surprisingly, protection occurred with concentrations of IAA (2-200 nM) much lower than those required to inhibit glycolysis. Pre-treatment with IAA results in suppression of oxyradical production and stabilization of mitochondrial function in neurons after exposure to oxidative insults. Levels of the stress heat-shock proteins HSP70 and HSP90, and of the anti-apoptotic protein Bcl-2, were increased in neurons exposed to IAA. Our data demonstrate that IAA can stimulate cytoprotective mechanisms within neurons, and suggest the possible use of IAA and related compounds in the prevention and/or treatment of neurodegenerative conditions.

Effects of myocardial ischemia and reperfusion on mitochondrial function and susceptibility to oxidative stress.

We investigated the effects of ischemia duration on the functional response of mitochondria to reperfusion and its relationship with changes in mitochondrial susceptibility to oxidative stress. Mitochondria were isolated from hearts perfused by the Langendorff technique immediately after different periods of global ischemia or reperfusion following such ischemia periods. Rates of O2 consumption and H2O2 release with complex I- and complex II-linked substrates, lipid peroxidation, overall antioxidant capacity, capacity to remove H2O2, and susceptibility to oxidative stress were determined. The effects of ischemia on some parameters were time dependent so that the changes were greater after 45 than after 20 min of ischemia, or were significantly different to the nonischemic control only after 45 min of ischemia. Thus, succinate-supported state 3 respiration exhibited a significant decrease after 20 min of ischemia and a greater decrease after 45 min, while pyruvate malate-supported respiration showed a significant decrease only after 45 min of ischemia, indicating an ischemia-induced early inhibition of complex II and a late inhibition of complex I. Furthermore, both succinate and pyruvate malate-supported H2O2 release showed significant increases only after 45 min of ischemia. Similarly, whole antioxidant capacity significantly increased and susceptibility to oxidants significantly decreased after 45 min of ischemia. Such changes were likely due to the accumulation of reducing equivalents, which are able to remove peroxides and maintain thiols in a reduced state. This condition, which protects mitochondria against oxidants, increases mitochondrial production of oxyradicals and oxidative damage during reperfusion. This could explain the smaller functional recovery of the tissue and the further decline of the mitochondrial function after reperfusion following the longer period of oxygen deprivation.

Progress in the Development of Caloric Restriction Mimetic Dietary Supplements

Caloric restriction (CR) - or reduced calorie intake with nutritional maintenance - can extend lifespan and increase resistance to age-related disease by mechanisms which likely involve reduced oxyradical production and cellular hormesis responses in which genes encoding cytoprotective proteins are upregulated. Recent studies suggest that several of the anti-aging effects of CR can be mimicked by giving animals compounds that reduce energy availability at the cellular level. Such CR mimetic compounds (e.g., 2-deoxy-D-glucose, phenformin and iodoacetate) have proven beneficial in experimental models of neurodegenerative disorders and cancer. CR mimetics appear to act via a hormesis-based mechanism in which cells upregulate heat-shock and other chaperone proteins, and growth factors. Although long-term effects of dietary supplementation with such CR mimetics remain to be determined, the initial findings reviewed here suggest a novel approach for the investigation of basic mechanisms of aging, for the possible extension of lifespan without CR, and for dealing with the rising tide of obesity in industrialized countries.

Selective and biphasic effect of the membrane lipid peroxidation product 4-hydroxy-2,3-nonenal on N-methyl-D-aspartate channels.

Increased oxyradical production and membrane lipid peroxidation occur in neurons under physiological conditions and in neurodegenerative disorders. Lipid peroxidation can alter synaptic plasticity and may increase the vulnerability of neurons to excitotoxicity, but the underlying mechanisms are unknown. We report that 4-hydroxy-2,3-nonenal (4HN), an aldehyde product of lipid peroxidation, exerts a biphasic effect on NMDA-induced current in cultured rat hippocampal neurons with current being increased during the first 2 h and decreased after 6 h. Similarly, 4HN causes an early increase and a delayed decrease in NMDA-induced elevation of intracellular Ca2+ levels. In contrast, 4HN affects neither the ion current nor the Ca2+ response to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). The initial enhancement of NMDA-induced current is associated with increased phosphorylation of the NR1 receptor subunit, whereas the delayed suppression of current is associated with cellular ATP depletion and mitochondrial membrane depolarization. Cell death induced by 4HN is attenuated by an NMDA receptor antagonist, but not by an AMPA receptor antagonist. A secreted form of amyloid precursor protein, previously shown to protect neurons against oxidative and excitotoxic insults, prevented each of the effects of 4HN including the early and late changes in NMDA current, delayed ATP depletion, and cell death. These findings show that the membrane lipid peroxidation product 4HN can modulate NMDA channel activity, suggesting a role for this aldehyde in physiological and pathophysiological responses of neurons to oxidative stress.

β-carotene at high concentrations induces apoptosis by enhancing oxy-radical production in human adenocarcinoma cells

This is the first report demonstrating a relationship between apoptosis induction and changes of intracellular redox potential in the growth-inhibitory effects of high concentrations of β-carotene in a tumor cell line. β-Carotene inhibited the growth of human WiDr colon adenocarcinoma cells in a dose- and time-dependent manner, induced apoptosis, and blocked Bcl-2 expression. These effects were accompanied by an enhanced production of intracellular reactive oxygen species (ROS). The addition of the antioxidant α-tocopherol blocked both the pro-oxidant and the growth-inhibitory effects of the carotenoid. These findings suggest that β-carotene may act as an inductor of apoptosis by its pro-oxidant properties.

On the true role of oxygen free radicals in the living state, aging, and degenerative disorders.

Oxyradicals are generally considered harmful byproducts of oxidative metabolism, causing molecular damage in living systems. They are implicated in various processes such as mutagenesis, aging, and series of pathological events. Although all this may be justified, evidence is accumulating that it is an oversimplified view of the real situation. We can assume nowadays that the living state of cells and organisms implicitly requires the production of oxyradicals. This idea is supported by experimental facts and arguments as follows. (1) Complete inhibition of the oxyradical production by KCN (or by any block of respiration) kills the living organisms much before the energy reserves would be exhausted. (2) Construction of the supramolecular organization of the cells (especially of their membranous compounds) requires the cross-linking effect of oxyradicals, particularly that of OH* radicals. (3) Blast type cells produce much fewer oxyradicals than do differentiated ones, and interventions increasing the production of OH* radicals induce differentiation of various lines of leukemic (HL-60 and K562) and normal (fibroblasts, chondroblasts, etc.) cells, while SOD expression increases greatly. (4) It is reasonable to assume that the continuous flux of OH* radicals is prerequisite to maintenance of constant electron delocalization on the proteins, which is a semiconductive phenomenon suggested in 1941 by Szent-Györgyi, but it has never been proven experimentally. It is theoretically possible to describe the function of the synapses as that of a single p-n-p transistor, assuming that the free radical flux maintains electron movements on the subsynaptic structures, while the actual membrane potential is governing the electron flux. This theoretical approach may open completely new possibilities for our understanding of the normal functions of living organisms, such as basic memory mechanisms in brain cells, their aging processes, and therapeutic approaches to many degenerative disorders, such as various types of dementia.

Induction of oxyradicals by arsenic: implication for mechanism of genotoxicity.

Although arsenic is a well-established human carcinogen, the mechanisms by which it induces cancer remain poorly understood. We previously showed arsenite to be a potent mutagen in human-hamster hybrid (A(L)) cells, and that it induces predominantly multilocus deletions. We show here by confocal scanning microscopy with the fluorescent probe 5',6'-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate that arsenite induces, within 5 min after treatment, a dose-dependent increase of up to 3-fold in intracellular oxyradical production. Concurrent treatment of cells with arsenite and the radical scavenger DMSO reduced the fluorescent intensity to control levels. ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOL-H) as a probe in conjunction with superoxide dismutase and catalase to quench superoxide anions and hydrogen peroxide, respectively, indicates that arsenite increases the levels of superoxide-driven hydroxyl radicals in these cells. Furthermore, reducing the intracellular levels of nonprotein sulfhydryls (mainly glutathione) in A(L) cells with buthionine S-R-sulfoximine increases the mutagenic potential of arsenite by more than 5-fold. The data are consistent with our previous results with the radical scavenger DMSO, which reduced the mutagenicity of arsenic in these cells, and provide convincing evidence that reactive oxygen species, particularly hydroxyl radicals, play an important causal role in the genotoxicity of arsenical compounds in mammalian cells.

Induction of oxyradicals by arsenic: Implication for mechanism of genotoxicity

Although arsenic is a well-established human carcinogen, the mechanisms by which it induces cancer remain poorly understood. We previously showed arsenite to be a potent mutagen in human-hamster hybrid (A(L)) cells, and that it induces predominantly multilocus deletions. We show here by confocal scanning microscopy with the fluorescent probe 5',6'-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate that arsenite induces, within 5 min after treatment, a dose-dependent increase of up to 3-fold in intracellular oxyradical production. Concurrent treatment of cells with arsenite and the radical scavenger DMSO reduced the fluorescent intensity to control levels. ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOL-H) as a probe in conjunction with superoxide dismutase and catalase to quench superoxide anions and hydrogen peroxide, respectively, indicates that arsenite increases the levels of superoxide-driven hydroxyl radicals in these cells. Furthermore, reducing the intracellular levels of nonprotein sulfhydryls (mainly glutathione) in A(L) cells with buthionine S-R-sulfoximine increases the mutagenic potential of arsenite by more than 5-fold. The data are consistent with our previous results with the radical scavenger DMSO, which reduced the mutagenicity of arsenic in these cells, and provide convincing evidence that reactive oxygen species, particularly hydroxyl radicals, play an important causal role in the genotoxicity of arsenical compounds in mammalian cells.

Neuroprotective signaling and the aging brain: take away my food and let me run

It is remarkable that neurons are able to survive and function for a century or more in many persons that age successfully. A better understanding of the molecular signaling mechanisms that permit such cell survival and synaptic plasticity may therefore lead to the development of new preventative and therapeutic strategies for age-related neurodegenerative disorders. We all know that overeating and lack of exercise are risk factors for many different age-related diseases including cardiovascular disease, diabetes and cancers. Our recent studies have shown that dietary restriction (reduced calorie intake) can increase the resistance of neurons in the brain to dysfunction and death in experimental models of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and stroke. The mechanism underlying the beneficial effects of dietary restriction involves stimulation of the expression of ‘stress proteins’ and neurotrophic factors. The neurotrophic factors induced by dietary restriction may protect neurons by inducing the production of proteins that suppress oxyradical production, stabilize cellular calcium homeostasis and inhibit apoptotic biochemical cascades. Interestingly, dietary restriction also increases numbers of newly-generated neural cells in the adult brain suggesting that this dietary manipulation can increase the brain’s capacity for plasticity and self-repair. Work in other laboratories suggests that physical and intellectual activity can similarly increase neurotrophic factor production and neurogenesis. Collectively, the available data suggest the that dietary restriction, and physical and mental activity, may reduce both the incidence and severity of neurodegenerative disorders in humans. A better understanding of the cellular and molecular mechanisms underlying these effects of diet and behavior on the brain is also leading to novel therapeutic agents that mimick the beneficial effects of dietary restriction and exercise.

Effects of heavy metals on phospholipase C in gill and digestive gland of the marine mussel Mytilus galloprovincialis Lam

We studied the in vivo and in vitro effects of Hg 2+ and Cu 2+ on the activity of phospholipase C (PLC), specific for phosphatidylinositol 4,5-bisphosphate, in the mussel ( Mytilus galloprovincialis Lam). The enzyme activity was assayed in tissue homogenates from gills and digestive gland. The toxic effect of Hg 2+ appeared to be stronger than that of Cu 2+ both in vitro and in vivo, especially for the digestive gland. In in vitro tests, Hg 2+ was able to inhibit PLC activity when added directly to the reaction mixture. Conversely, Cu 2+ was effective only after preincubation, suggesting that the effect of the metal may be derived from lipid peroxidation due to Cu 2+-induced oxyradical production. Treatment of mussels with sublethal concentrations of Hg 2+ or Cu 2+ in vivo produced significant PLC inhibition after 1 or 4 days, respectively. A recovery was reached after 7 days of in vivo metal incubation. Data indicate that in mussel gills and digestive gland heavy metals impair PLC activity, thereby affecting IP 3-dependent Ca 2+ signaling.

Starch-deferoxamine conjugate inhibits hepatocyte Ca2+ uptake during hemorrhagic shock and resuscitation.

This study investigated whether hepatocyte Ca2+ dysregulation after hemorrhagic shock and resuscitation could be modulated by the iron chelator hydroxyethyl starch-conjugated deferoxamine (HES-DFO). In a randomized experimental study, anesthetized rats (n = 7) were bled for 60 minutes to maintain mean arterial blood pressure at 40 mm Hg. They were then resuscitated with 60% of shed blood and threefold the shed-blood volume as lactated Ringer's solution, 1 mL of pentastarch solution (hydroxyethyl starch 10%) per mL of shed blood, or 1 mL of HES-DFO solution (10%) per mL of shed blood. In isolated hepatocytes, the rate of Ca2+ influx (Ca2+ in), total Ca2+ uptake (Ca2+ up), and membrane Ca2+ flux (Ca2+ flux) were determined by 45Ca incubation. Reduced or oxidized glutathione and malondialdehyde concentrations were assessed fluorometrically. Significant increases of hepatocellular Ca2+ in, Ca2+ up, and Ca2+ flux were observed in rats resuscitated with lactated Ringer's solution compared with control groups (p < 0.05). Although hydroxyethyl starch decreased Ca2+ in but not Ca2+ up, HES-DFO not only prevented the increase of Ca2+ in and Ca2+ up but also inhibited hepatocyte oxidative injury. Iron-catalyzed oxyradical production and membrane peroxidation seem to alter hepatocyte Ca2+ homeostasis after hemorrhagic shock and resuscitation.

Mitochondrial GPx1 Decreases Induced but Not Basal Oxidative Damage to mtDNA in T47D Cells

The production of oxyradicals by mitochondria (mt) is a source of oxidative damage to mtDNA such as 8-oxo-dG lesions that may lead to mutations and mitochondrial dysfunction. The potential protection of mtDNA by glutathione peroxidase-1 (GPx1) was investigated in GPx1-proficient (GPx-2) and GPx1-deficient (Hygro-3) human breast T47D cell transfectants. GPx activity and GPx1-like antigen concentration in mitochondria were respectively at least 100-fold and 20- to 25-fold higher in GPx2 than Hygro-3 cells. In spite of this large difference in peroxide-scavenging capacity, the basal 8-oxo-dG frequency in mtDNA, assessed by carefully controlled postlabeling assay, was strikingly similar in both cell lines. In contrast, in response to menadione-mediated oxidative stress, induction of 8-oxo-dG and DNA strand breaks was much lower in the GPx1-proficient mitochondria (e.g., +14% 8-oxo-dG versus +54% in Hygro-3 after 1-h exposure to 25 μM menadione, P < 0.05). Our data indicate that the mitochondrial glutathione/GPx1 system protected mtDNA against damage induced by oxidative stress, but did not prevent basal oxidative damage to mtDNA, which, surprisingly, appeared independent of GPx1 status in the T47D model.

Iron Ore Mines Leachate Potential for Oxyradical Production

The ecotoxicological effects of mining effluents is coming under much greater scrutiny. It appears necessary to explore possible health effects in association with iron ore mining effluents. The present results clearly demonstrate that iron-ore leachate is not an inert media but has the potential to induce lipid peroxidation. Peroxidation was assessed by measuring oxygen consumption in the presence of a reducing agent such as ascorbate or NADPH and a chelator such as EDTA. Labrador iron ore is an insoluble complex crystalline material containing a mixture of metals (Fe, Al, Ti, Mn, Mg,…, ) in contrast to the iron sources used for normal lipid peroxidation studies. The metal of highest percentage is iron (59.58%), a metal known to induce oxyradical production. Iron ore powder initiated ascorbic acid-dependent lipid peroxidation (nonenzymatic) in liposomes, lipids extracted from rat and salmon liver microsomes, and intact salmon liver microsomes. It also revealed an inhibitory effect of NADPH-dependent microsomes lipid peroxidation as well as on NADPH cytochrome c reductase activity. However, nonenzymatic peroxidation in rat liver microsomes was not significantly inhibited. Cytochrome P450 IA1- and IIB1-dependent enzymatic activities as well as P450 levels were not affected. The inhibition could be due to one of the other components of iron ore leachate (Mn, Al,…, ). These effects of iron-ore leachate indicate that a potential toxicity could be associated with its release into lakes. Further studies are necessary to explore in vivo effects on aquatic animals.

Apoptotic and anti-apoptotic synaptic signaling mechanisms.

Although several prominent morphological features of apoptosis are evident in the cell body (e.g., cell shrinkage, membrane blebbing, and nuclear DNA condensation and fragmentation) the biochemical and molecular cascades that constitute the cell death machinery can be engaged in synaptic terminals and neurites. Initiating events such as oxyradical production and calcium influx, and effector processes such as Par-4 production, mitochondrial alterations and caspase activation, can be induced in synapses and neurites. Several prominent signal transduction pathways in synaptic terminals play important roles in either promoting or preventing neuronal death in physiological and pathological conditions. For example, activation of glutamate receptors in postsynaptic spines can induce neuronal apoptosis, whereas local activation of neurotrophic factor receptors in presynaptic terminals can prevent neuronal death. Factors capable of inducing nuclear chromatin condensation and fragmentation can be produced locally in synaptic terminals and neurites, and may propogate to the cell body. Recent findings suggest that, beyond their roles in inducing or preventing cell death, apoptotic and anti-apoptotic cascades play roles in synaptic plasticity (structural remodelling and long-term functional changes). For example, caspase activation results in proteolysis of glutamate receptor (AMPA) subunits, which results in altered neuronal responsivity to glutamate. Activation of neurotrophic factor receptors in synaptic terminals can result in local changes in energy metabolism and calcium homeostasis, and can induce long-term changes in synaptic transmission. The emerging data therefore suggest that synapses can be considered as autonomous compartments in which both pro- and anti-apoptotic signaling pathways are activated resulting in structural and functional changes in neuronal circuits. A better understanding of such synaptic signaling mechanisms may reveal novel approaches for preventing and treating an array of neurodegenerative conditions that are initiated by perturbed synaptic homeostasis.

Roles of nuclear factor kappaB in neuronal survival and plasticity.

The transcription factor nuclear factor kappaB (NF-kappaB) is moving to the forefront of the fields of apoptosis and neuronal plasticity because of recent findings showing that activation of NF-kappaB prevents neuronal apoptosis in various cell culture and in vivo models and because NF-kappaB is activated in association with synaptic plasticity. Activation of NF-kappaB was first shown to mediate antiapoptotic actions of tumor necrosis factor in cultured neurons and was subsequently shown to prevent death of various nonneuronal cells. NF-kappaB is activated by several cytokines and neurotrophic factors and in response to various cell stressors. Oxidative stress and elevation of intracellular calcium levels are particularly important inducers of NF-kappaB activation. Activation of NF-kappaB can interrupt apoptotic biochemical cascades at relatively early steps, before mitochondrial dysfunction and oxyradical production. Gene targets for NF-kappaB that may mediate its antiapoptotic actions include the antioxidant enzyme manganese superoxide dismutase, members of the inhibitor of apoptosis family of proteins, and the calcium-binding protein calbindin D28k. NF-kappaB is activated by synaptic activity and may play important roles in the process of learning and memory. The available data identify NF-kappaB as an important regulator of evolutionarily conserved biochemical and molecular cascades designed to prevent cell death and promote neuronal plasticity. Because NF-kappaB may play roles in a range of neurological disorders that involve neuronal degeneration and/or perturbed synaptic function, pharmacological and genetic manipulations of NF-kappaB signaling are being developed that may prove valuable in treating disorders ranging from Alzheimer's disease to schizophrenia.

AT1 receptor antagonism enhances angiotensin-II-facilitated carrageenan-induced paw edema.

Recent studies have demonstrated that the renin-angiotensin system (RAS) participates in the processes of inflammation. An active component of this system, angiotensin II (Ang II), differentially regulates the production of oxyradicals, nitric oxide, prostaglandins, platelet-activating factors and bradykinins by acting through AT1 or AT2 receptor subtypes. Many of the physiological actions of Ang II mediated through AT1 and AT2 receptors are opposite and thereby show physiological antagonism to each other. In the present study, we evaluated the effect of locally administered Ang II, the ACE inhibitor captopril and the AT1 receptor antagonist losartan in the carrageenan model of acute inflammation. Local administration of losartan (10-50 micrograms/paw) or Ang II (0.2-1 microgram/paw) alone did not induce inflammation, but significantly enhanced the carrageenan-induced edema in a dose-dependent manner. Coadministration of subeffective doses of losartan (10 micrograms/paw) and Ang II (0.2 microgram/paw) significantly potentiated the carrageenan-induced inflammation. In conclusion, the present study predicts that Ang II might be formed locally during carrageenan-induced acute inflammation. Potentiation of the Ang II effect in carrageenan-induced inflammation by losartan may be mediated through over-stimulation of unblocked AT2 receptors or due to stimulation of inflammatory pathways by unknown mechanisms.

DNA strand scission by the nephrotoxin [2,2′-bipyridine]-3,3′,4,4′-tetrol-1,1′-dioxide and related compounds in the presence of iron

The capacity of non-illuminated nephrotoxin orellanine ([2,2′-bipyridine]-3,3′,4,4′-tetrol-1,1′-dioxide) to induce DNA damage in the presence of ferrous iron and dioxygen has been evaluated. Maximal single-strand breaks in plasmid DNA were obtained with a metal to ligand ratio 1:3. Instantaneous oxidation of Fe2+ in presence of orellanine under air was responsible for oxy-radical production concomitant to a stable ferric complex Fe(III)Or3 formation, leading to oxidative DNA breakage at physiological pH. DNA damage was lowered in the presence of SOD and catalase or DMSO, indicating a set of reactions that leads to oxyradical generation. Iron chelators such as DTPA and EDTA had no protecting effect, Desferal slightly protected. GSH acted as an oxy-radical scavenger, whereas cysteine induced stronger damage.Closely related bipyridine compounds were also studied in presence of Fe2+ and O2 using a combination of spin-trapping and DNA-nicking experiments, none of which were able to chelate iron and induce damage at pH 7. Both catecholic moieties and aminoxide groups are required for observing breakage at physiological pH.

Role of metallothionein against oxidative stress in the mussel Mytilus galloprovincialis.

Metallothionein (MT) is a sulfhydryl-rich protein involved mainly in heavy metal homeostasis and detoxification. In this study, the use of the mussel as an experimental model allowed us to test MT antioxidant properties at the molecular, cellular, and organism level. MT induction was achieved by mussel exposure to Cd (200 microg/l) in aquaria for 7 days followed by detoxification in the sea for 28 days. Cd-preexposed and nonexposed mussels were then treated with Fe (300-600 microg/l) in aquaria for 3 days. Biochemical assays on digestive gland tissue showed that treatment with Fe led to a significant increase in oxyradical production and malondialdehyde level only in mussels not preexposed to Cd. The Cd-dependent resistance to oxidative stress was ascribed to MT induction, as Cd produced no significant variation of reduced glutathione and major antioxidant enzymes. Digital imaging of isolated digestive gland cells showed lower oxyradical rise and higher viability in cells from Cd-preexposed mussels after treatments with 0.5-5 mM H2O2. Analyses on whole organisms showed that anoxic survival was lowered in mussels that had been treated with Fe, but such an effect was less pronounced in Cd-preexposed mussels compared with nonpreexposed ones. In conclusion, data suggest an antioxidant role for MT, which seems to occur through oxyradical scavenging and is able to protect both isolated cells and the entire organism from oxidative stress.