Brain oxidative stress in rat with chronic iron or copper overload.

Male rats of 80-90 g that were fed 42 days with a commercial rodent diet of 2780 kcal/100 g and received chronic overloads of either Fe(II) or Cu(II) in the drinking water. The two metals produced brain oxidative stress and damage with marked increases in the indicators of oxidative processes: in vivo brain surface chemiluminescence (the sensitive organ non-invasive assay for oxidative free radical reactions), and the ex vivo processes of phospholipid peroxidation and protein oxidation. Brain redox imbalance was also indicated by marked decreases in the cellular indicators of oxidative metabolic stress: reduced glutathione (GSH) content and reduced/oxidized glutathione ratio (GSH/GSSG). Brain decreased GSH content has a central role in the biochemical oxidative processes associated with Fe and Cu chronic damage. The understanding of biochemical oxidative imbalances in the rat brain with chronic Fe(II) or Cu(II) overloads may be useful for the establishment of pharmacological therapies for human pathologies associated to Fe and Cu cellular imbalances.

Redox dyshomeostasis in the experimental chronic hepatic overloads with iron or copper.

Male rats of 80-90 g were overloaded with either Fe(II) or Cu(II) for 42 days by high concentrations of FeCl2 or CuSO4 in the drinking water. The animals were fed with a commercial rodent diet of 2780 kcal/100 g. Both metal treatments led to a liver redox imbalance and dyshomeostasis with oxidative stress and damage and the concomitant enhancement of oxidative processes as indicated by in vivo surface liver chemiluminescence, the sensitive and organ non-invasive assay for oxidative free radical reactions, and by ex vivo determined processes of phospholipid peroxidation and protein oxidation. In parallel, marked decreases in the antioxidant defense were observed. Liver reduced glutathione (GSH) content and the reduced/oxidized glutathione ratio (GSH/GSSG) were early indicators of oxidative metabolic disturbance upon the metal overloads. Thus, GSH plays a central role in the defense reactions involved in the chronic toxicity of Fe and Cu. Chronic overloads of Fe or Cu in rats afford an experimental animal model of hemochromatosis and of Wilson's disease, respectively. These two animal models could be useful in the study and development of the beneficial effects of pharmacological interventions in the two human diseases.

Mitochondrial dysfunction as a mediator of hippocampal apoptosis in a model of hepatic encephalopathy.

In this study, we describe the presence of apoptosis, associated with a mitochondrial dysfunction in the hippocampus of animals in an experimental model defined as minimal hepatic encephalopathy (MHE). This experimental model was studied after 10 days of induced portal vein calibrated stricture, leading to portal hypertension and to a moderate hyperammonemia, without the presence of other evident central nervous system changes. The molecular mechanisms here proposed indicate the presence of apoptotic intrinsic pathways that point to hippocampal mitochondria as an important mediator of apoptosis in this experimental model. In this model of MHE, the presence of DNA fragmentation is documented by 2.3-times increased number of TUNEL-positive cells. These findings together with a higher ratio of the Bcl-2 family members Bax/Bcl-xL in the outer mitochondrial membrane of the MHE animals together with 11% of cytochrome c release indicate the presence of apoptosis in this experimental model. A detailed analysis of the hippocampal mitochondrial physiology was performed after mitochondrial isolation. The determination of the respiratory rate in the presence of malate plus glutamate and ADP showed a 45% decrease in respiratory control in MHE animals as compared with the sham group. A marked decrease of cytochrome oxidase (complex IV of the electron transport chain) was also observed, showing 46% less activity in hippocampal mitochondria from MHE animals. In addition, mitochondria from these animals showed less ability to maintain membrane potential (ΔΨ (m)) which was 13% lower than the sham group. Light scattering experiments showed that mitochondria from MHE animals were more sensitive to swell in the presence of increased calcium concentrations as compared with the sham group. In addition, in vitro studies performed in mitochondria from sham animals showed that mitochondrial permeability transition (MPT) could be a mitochondrial mediator of the apoptotic signaling in the presence of NH(4) (+) and calcium.

Nitric oxide and superoxide anion production during heparin-induced capacitation in cryopreserved bovine spermatozoa.

The aim of this work was to quantify NO,O(2)(-) and ONOO(-) production during heparin-induced capacitation of cryopreserved bovine spermatozoa. A time dependent hyperbolic increase was observed for heparin-dependent capacitation, O(2) uptake, and NO production. Conversely, O(2)(-) production was increased during the first 15 min of incubation, showing a decrease from this time until 45 min. At 15 min of heparin incubation, a threefold increase in O(2) consumption (5.9 ± 0.6 nmol/min × 10(7) cells), an enhancement in NO release (1.1 ± 0.2 nmol/min × 10(7) cells), and a five-fold increase in O(2)(-) production (1.3 ± 0.07 nmol/min × 10(7) cells), were observed. Peroxynitrite production rate was estimated taking into account NO and O(2)(-) generation and the second-order rate constant of the reaction between these species. To conclude, heparin-induced capacitation of cryopreserved bovine spermatozoa activates (i) mitochondrial O(2) uptake by high ADP levels due to increased energy requirements, (ii) NO production by a constitutive NOS and (iii) O(2)(-) production by a membrane-bound NAD(P)H oxidase. The products of both enzymes are released to the extracellular space and could be involved in the process of sperm capacitation.

Bioactivity of sesquiterpenes: compounds that protect from alcohol-induced gastric mucosal lesions and oxidative damage.

Sesquiterpene lactones of the guaianolide and eudermanolide types are considered of interest because they have an effect in the regulation and prevention of oxidative damage and inflammation-mediated biological damage. Dehydroleucodine, a natural sesquiterpene isolated from Artemisia douglasiana Besser, and ilicic aldehyde, a semi-synthetic sesquiterpene lactones, showed in vivo protection in ethanol-induced gastric mucosa damage. This action was determined by in situ gastric mucosa chemiluminescence and by tissue antioxidant content.

Antioxidant capacity of a 3-deoxyanthocyanidin from soybean.

Soybean cotyledons directly exposed to UV-C (190-280 nm) contained a colored pigment in those areas of the epidermis directly exposed to UV-C. Ethanolic extracts from UV-C irradiated cotyledons showed a significant peak at 532 nm at pH=10, but not seen at pH=6, successive changes in pH were accompanied by reversible changes in the spectra. The identity of the pigment isolated from soybean cotyledons was established as apigeninidin by comparing the features of standard of a apigeninidin (from sorghum) previously characterized by FAB-MS, UV, HPLC, 1H NMR, and IR spectroscopy. To characterize antioxidant activity of this compound, its ability to scavenge radical species in vitro was tested. In the concentration range tested (up to 200 microg ml (-1)), apigeninidin did not show any scavenger activity towards hydroxyl radical, quinones or NO. However, ascorbyl radical and lipid radicals were effectively quenched in a dose-dependent manner. Overall, UV-C radiation triggers molecular signals that lead in soybean cotyledons to the synthesis and accumulation of an antioxidant pigment, apigeninidin, that shows scavenger activity against ascorbyl and lipid radicals in in vitro studies.

Nitric oxide inhibits mitochondrial NADH:ubiquinone reductase activity through peroxynitrite formation.

This study was aimed at assessing the effects of long-term exposure to NO of respiratory activities in mitochondria from different tissues (with different ubiquinol contents), under conditions that either promote or prevent the formation of peroxynitrite. Mitochondria and submitochondrial particles isolated from rat heart, liver and brain were exposed either to a steady-state concentration or to a bolus addition of NO. NO induced the mitochondrial production of superoxide anions, hydrogen peroxide and peroxynitrite, the latter shown by nitration of mitochondrial proteins. Long-term incubation of mitochondrial membranes with NO resulted in a persistent inhibition of NADH:cytochrome c reductase activity, interpreted as inhibition of NADH:ubiquinone reductase (Complex I) activity, whereas succinate:cytochrome c reductase activity, including Complex II and Complex III electron transfer, remained unaffected. This selective effect of NO and derived species was partially prevented by superoxide dismutase and uric acid. In addition, peroxynitrite mimicked the effect of NO, including tyrosine nitration of some Complex I proteins. These results seem to indicate that the inhibition of NADH:ubiquinone reductase (Complex I) activity depends on the NO-induced generation of superoxide radical and peroxynitrite and that Complex I is selectively sensitive to peroxynitrite. Inhibition of Complex I activity by peroxynitrite may have critical implications for energy supply in tissues such as the brain, whose mitochondrial function depends largely on the channelling of reducing equivalents through Complex I.

Nitric oxide and superoxide radical production by human mononuclear leukocytes.

Human mononuclear cells (90% lymphocytes, 9% monocytes, and 1% polymorphonuclear leukocytes) produced spontaneously in resting state 0.11+/-0.01 nmol of nitric oxide (NO)/min/10(6) cells and 0.25+/-0.02 nmol of superoxide anion (O2-)/min/10(6) cells, as primary products. When these cells were stimulated with phorbol 12-myristate 13-acetate (PMA), the NO and O2- production increased by 82% and 204% to 0.25+/-0.02 nmol of NO/min/10(6) cells and 0.76+/-0.12 nmol of O2-/min/10(6) cells, respectively. Oxygen uptake reasonably accounted for the sum of the rates of NO and hydrogen peroxide (H2O2), the latter calculated as 0.5 O2- production, in nonstimulated and in PMA-stimulated cells. H2O2 and peroxynitrite formation were detected and measured as secondary products of the primary products O2- and NO. An original assay to determine H2O2 steady-state concentration and production rates is described. The determined production rates of the involved reactive species are in good agreement with known chemical equations. It is apparent that NO and O2- production by human mononuclear cells may constitute the basis of intercellular signaling and cell toxicity.

Oxidative stress and mitochondrial damage in coronary artery bypass graft surgery: effects of antioxidant treatments.

We examined antioxidant actions in 73 patients undergoing coronary artery surgery by assessing mitochondrial damage and oxidative stress in ventricular biopsies obtained at preischemia and postreperfusion. Those patients who received antioxidant therapy benefited by less oxidative stress and mitochondrial damage.

Reactions of peroxynitrite in the mitochondrial matrix.

Superoxide radical (O2-) and nitric oxide (NO) produced at the mitochondrial inner membrane react to form peroxynitrite (ONOO-) in the mitochondrial matrix. Intramitochondrial ONOO- effectively reacts with a few biomolecules according to reaction constants and intramitochondrial concentrations. The second-order reaction constants (in M(-1) s(-1)) of ONOO- with NADH (233 +/- 27), ubiquinol-0 (485 +/- 54) and GSH (183 +/- 12) were determined fluorometrically by a simple competition assay of product formation. The oxidation of the components of the mitochondrial matrix by ONOO- was also followed in the presence of CO2, to assess the reactivity of the nitrosoperoxocarboxylate adduct (ONOOCO2-) towards the same reductants. The ratio of product formation was about similar both in the presence of 2.5 mM CO2 and in air-equilibrated conditions. Liver submitochondrial particles supplemented with 0.25-2 microM ONOO- showed a O2- production that indicated ubisemiquinone formation and autooxidation. The nitration of mitochondrial proteins produced after addition of 200 microM ONOO- was observed by Western blot analysis. Protein nitration was prevented by the addition of 50-200 microM ubiquinol-0 or GSH. An intramitochondrial steady state concentration of about 2 nM ONOO- was calculated, taking into account the rate constants and concentrations of ONOO- coreactants.

Content of liver and brain ubiquinol-9 and ubiquinol-10 after chronic ethanol intake in rats subjected to two levels of dietary alpha-tocopherol.

To assess the effect of chronic ethanol ingestion in the content of the reduced forms of coenzymes Q9 (ubiquinol-9) and Q10 (ubiquinol-10) as a factor contributing to oxidative stress in liver and brain, male Wistar rats were fed ad libitum a basal diet containing either 10 or 2.5 mg alpha-tocopherol/100 g diet (controls), or the same basal diet plus a 32% ethanol-25% sucrose solution. After three months treatment, ethanol chronically-treated rats showed identical growth rates to the isocalorically pair-fed controls, irrespectively of alpha-tocopherol dietary level. Lowering dietary alpha-tocopherol led to a decreased content of this vitamin in the liver and brain of control rats, without changes in that of ubiquinol-9, and increased levels of hepatic ubiquinol-10 and total glutathione (tGSH), accompanied by a decrease in brain tGSH. At the two levels of dietary alpha-tocopherol, ethanol treatment significantly decreased the content of hepatic alpha-tocopherol and ubiquinols 9 and 10. This effect was significantly greater at 10 mg alpha-tocopherol/100 g diet than at 2.5, whereas those of tGSH were significantly elevated by 43% and 9%, respectively. Chronic ethanol intake did not alter the content of brain alpha-tocopherol and tGSH, whereas those of ubiquinol-9 were significantly lowered by 20% and 14% in rats subjected to 10 and 2.5 mg alpha-tocopherol/100 g diet, respectively. It is concluded that chronic ethanol intake at two levels of dietary alpha-tocopherol induces a depletion of hepatic alpha-tocopherol and ubiquinols 9 and 10, thus contributing to ethanol-induced oxidative stress in the liver tissue. This effect of ethanol is dependent upon the dietary level of alpha-tocopherol, involves a compensatory enhancement in hepatic tGSH availability, and is not observed in the brain tissue, probably due to its limited capacity for ethanol biotransformation and glutathione synthesis.

Antioxidant adaptive response of human mononuclear cells to UV-B: effect of lipoic acid.

Supplementation of human mononuclear cells with 3 and 6 mM of lipoic acid produces an inhibition of the antioxidant adaptive response triggered by treatment with UV-B light (0.30 W/m2 for 15 min). Supplementation with 1.5 mM of lipoic acid gives no conclusive results. The adaptive response is characterized by an increase in the activities of superoxide dismutase, catalase, glutathione peroxidase and DT-diaphorase. Catalase (5.5 +/- 0.6 pmol/mg prot) increases its activity by up to 22 +/- 3 pmol/mg prot, after irradiation with UV-B. Supplementation with 3 and 6 mM of lipoic acid completely inhibits the adaptive response. The activities of the membrane-bound mitochondrial enzymes succinate dehydrogenase and cytochrome oxidase do not increase after UV-B exposure. Moreover, their activities are found to decrease and the addition of lipoic acid does not prevent this effect. The inhibition of the antioxidant response by lipoic acid in human cells appears as indirect evidence of the existence of oxidative stress in the development of this response. As lipoic acid behaves as an effective antioxidant, it seems that its action decreases the intracellular oxidative signals necessary to develop the adaptive response in human mononuclear cells.

Regulation of mitochondrial respiration by oxygen and nitric oxide.

Although the regulation of mitochondrial respiration and energy production in mammalian tissues has been exhaustively studied and extensively reviewed, a clear understanding of the regulation of cellular respiration has not yet been achieved. In particular, the role of tissue pO2 as a factor regulating cellular respiration remains controversial. The concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers has evolved in the last few years and is still being researched. A recent concept that regulation of cellular respiration is regulated by ADP, O2 and NO preserves the notion that energy demands drive respiration but places the kinetic control of both respiration and energy supply in the availability of ADP to F1-ATPase and of O2 and NO to cytochrome oxidase. In addition, recent research indicates that NO participates in redox reactions in the mitochondrial matrix that regulate the intramitochondrial steady state concentration of NO itself and other reactive species such as superoxide radical (O2-) and peroxynitrite (ONOO-). In this way, NO acquires an essential role as a mitochondrial regulatory metabolite. No exhibits a rich biochemistry and a high reactivity and plays an important role as intercellular messenger in diverse physiological processes, such as regulation of blood flow, neurotransmission, platelet aggregation and immune cytotoxic response.

Oxidation of ubiquinol by peroxynitrite: implications for protection of mitochondria against nitrosative damage.

A major pathway of nitric oxide utilization in mitochondria is its conversion to peroxynitrite, a species involved in biomolecule damage via oxidation, hydroxylation and nitration reactions. In the present study the potential role of mitochondrial ubiquinol in protecting against peroxynitrite-mediated damage is examined and the requirements of the mitochondrial redox status that support this function of ubiquinol are established. (1) Absorption and EPR spectroscopy studies revealed that the reactions involved in the ubiquinol/peroxynitrite interaction were first-order in peroxynitrite and zero-order in ubiquinol, in agreement with the rate-limiting formation of a reactive intermediate formed during the isomerization of peroxynitrite to nitrate. Ubiquinol oxidation occurred in one-electron transfer steps as indicated by the formation of ubisemiquinone. (2) Peroxynitrite promoted, in a concentration-dependent manner, the formation of superoxide anion by mitochondrial membranes. (3) Ubiquinol protected against peroxynitrite-mediated nitration of tyrosine residues in albumin and mitochondrial membranes, as suggested by experimental models, entailing either addition of ubiquinol or expansion of the mitochondrial ubiquinol pool caused by selective inhibitors of complexes III and IV. (4) Increase in membrane-bound ubiquinol partially prevented the loss of mitochondrial respiratory function induced by peroxynitrite. These findings are analysed in terms of the redox transitions of ubiquinone linked to both nitrogen-centred radical scavenging and oxygen-centred radical production. It may be concluded that the reaction of mitochondrial ubiquinol with peroxynitrite is part of a complex regulatory mechanism with implications for mitochondrial function and integrity.

Nitric oxide production and mitochondrial dysfunction during rat thymocyte apoptosis.

Production of nitric oxide (NO) by mitochondrial membranes as methemoglobin formation sensitive to N(G)-methyl-l-arginine inhibition and mitochondrial O(2) consumption in metabolic states 3 and 4 and the respiratory control (state 3/state 4) were measured at early stages of rat thymocyte apoptosis. Programmed cell death was induced by addition of methylprednisolone and etoposide to thymocyte suspensions. Increased NO production by mitochondrial membranes was observed after 30 min of methylprednisolone and etoposide addition and was accompanied by mitochondrial respiratory impairment as an early phenomenon in apoptotic thymocytes. The respiratory control in isolated mitochondria from untreated thymocytes was 4.2 +/- 0.2 and decreased to 3.1 +/- 0.2 and 1.9 +/- 0.3 after 1 h of methylprednisolone and etoposide treatment, respectively. The low mitochondrial respiratory control was accompanied by a marked decrease in GSH and cytochrome c content. Moreover, an inhibitory effect in the amount of apoptosis due to thymocyte pretreatment with N(G)-methyl-l-arginine and N(omega)-nitro-(l)-arginine (l-NNA), indicate that nitric oxide production is closely involved in the signaling of rat thymocyte apoptosis.

In situ determination by surface chemiluminescence of temporal relationships between evolving warm ischemia-reperfusion injury in rat liver and phagocyte activation and recruitment.

Liver ischemia-reperfusion is characterized by an increased oxygen-dependent free radical chain-reaction rate and an increased steady-state concentration of reactive oxygen species. The aim of this study was to evaluate the in situ generation of reactive oxygen species and its relationship with phagocyte activation and recruitment in reperfused rat liver. Rat livers were subjected to 2 hours of selective lobular ischemia and reperfusion for up to 12 hours. The following parameters were determined: in situ liver chemiluminescence, understood to reflect the tissue steady-state concentration of singlet oxygen ((1)O(2)); myeloperoxidase tissue activity; the number of neutrophils; and the degree of necrosis. An early chemiluminescence burst was measured after 30 minutes of blood reflow (early phase of oxidative stress), followed by a relapse and a further increase after 4 to 12 hours of reperfusion (late phase of oxidative stress). Both early and late phases were modified by pretreatment with gadolinium chloride (GdCl(3)), pointing to a key role of the Kupffer cells. Neutrophils infiltrated into the liver, myeloperoxidase activity, in situ chemiluminescence, and necrosis were found to be strongly correlated over the 4- to 12-hour reperfusion period (r =.960; average of the 4 correlation coefficients). Together with resident phagocytes, neutrophil recruitment and activation appear to provide a major contribution to the increase of oxygen-dependent free-radical reactions and amplification of liver reperfusion damage. Surface chemiluminescence appears to properly describe the in situ and in vivo progressive organization of the acute inflammatory response with phagocyte-mediated liver injury.

Contribution of gamma glutamyl transpeptidase to oxidative damage of ischemic rat kidney.

BACKGROUND: A variety of mechanisms have been considered in the pathogenesis of the cell damage occurring in the kidney that is undergoing transient ischemia. However, little information is available about the role of oxidative stress in building up the tissue injury in the hypoxic organ during short-term ischemia. METHODS: After a standard brief period (25 min) of unilateral kidney ischemia in rats, pretreated or not with acivicin (60 micromol/L/kg i.v.), tissue samples from both ischemic and not ischemic kidneys were obtained to measure malondialdehyde (MDA) and glutathione (GSH) content, gamma glutamyl transpeptidase (GGT) activity by spectrophotometry, localization and intensity of enzyme activity, and tissue damage by histochemistry. RESULTS: GGT activity was found to be increased in both cortical and medullar zones of the ischemic kidneys, where the GSH level was only slightly decreased and the MDA level, in contrast, was markedly increased; in parallel, the cytosolic volume of the proximal tubular (PT) cells showed a significant increment. The animal pretreatment with acivicin, a specific inhibitor of GGT, besides preventing the up-regulation of the enzyme during ischemia, afforded good protection against the observed changes of MDA and GSH tissue levels, as well as of tubular cell volume. CONCLUSIONS: Ex vivo data supporting a net pro-oxidant effect of up-regulated GGT during short-term ischemia of rat kidney have been obtained. The enzyme stimulation appears to contribute to the renal morphological damage exerted by a brief hypoxic condition at the level of PT cells. The actual impact on kidney function by GGT-dependent oxidative damage during transient ischemia and the potential protective action of GGT inhibitors require subsequent investigation.

Analysis of the pathways of nitric oxide utilization in mitochondria.

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen- and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.

Free radical chemistry in biological systems.

Mitochondria are an active source of the free radical superoxide (O2-) and nitric oxide (NO), whose production accounts for about 2% and 0.5% respectively, of mitochondrial O2 uptake under physiological conditions. Superoxide is produced by the auto-oxidation of the semiquinones of ubiquinol and the NADH dehydrogenase flavin and NO by the enzymatic action of the nitric oxide synthase of the inner mitochondrial membrane (mtNOS). Nitric oxide reversibly inhibits cytochrome oxidase activity in competition with O2. The balance between NO production and its utilization results in a NO intramitochondrial steady-state concentration of 20-50 nM, which regulates mitochondrial O2 uptake and energy supply. The regulation of cellular respiration and energy production by NO and its ability to switch the pathway of cell death from apoptosis to necrosis in physiological and pathological conditions could take place primarily through the inhibition of mitochondrial ATP production. Nitric oxide reacts with O2- in a termination reaction in the mitochondrial matrix, yielding peroxynitrite (ONOO-), which is a strong oxidizing and nitrating species. This reaction accounts for approximately 85% of the rate of mitochondrial NO utilization in aerobic conditions. Mitochondrial aging by oxyradical- and peroxynitrite-induced damage would occur through selective mtDNA damage and protein inactivation, leading to dysfunctional mitochondria unable to keep membrane potential and ATP synthesis.