Effect of Short-Term Caloric Restriction on H2O2 Production and Oxidative DNA Damage in Rat Liver Mitochondria and Location of the Free Radical Source

Oxygen free radicals (ROS) of mitochondrial origin seem to be involved in aging. Whereas in other tissues complexes I or III of the respiratory chain contain the ROS generators, in this study we find that rat liver mitochondria generate oxygen radicals at complexes I, II, and III. Short-term (6 weeks) caloric restriction significantly decreased H₂O₂ production in rat liver mitochondria. This decrease in ROS production was located at complex I because it occurred with complex I-linked substrates (pyruvate/malate), but did not reach statistical significance with the complex II-linked substrate succinate. The mechanism responsible for the lowered ROS production was not a decrease in oxygen consumption. Instead, the mitochondria of caloric-restricted animals released less ROS per unit electron flow. This was due to a decrease in the degree of reduction of the complex I generator. Furthermore, oxidative damage to mitochondrial and nuclear DNA was also decreased in the liver by short-term caloric restriction. The results agree with the idea that caloric restriction delays aging, at least in part, by decreasing the rate of mitochondrial ROS generation and thus the rate of attack to molecules, like DNA, highly relevant for the accumulation of age-dependent changes.     

The Control of Iron-Induced Oxidative Damage in Isolated Rat-Liver Mitochondria by Respiration State and Ascorbate

The reaction of iron (II) with H₂O₂ is believed to generate highly reactive species (e.g., OH) capable of initiating biological damage. This study investigates the possibility that the severity of oxidative damage induced by iron in hepatic mitochondria is determined by the level of mitochondrial-H₂O₂ generation, which is believed to be particularly prominent in state-4 respiration.

Iron-induced damage is found to be greater in state-4 than in state-3 respiration. Experiments using uncoupling agents and Ca⁺⁺ to mimic state-3 conditions indicate that this effect reflects differences in the steady-state oxidation-level of the electron carriers of the respiratory chain (and hence the level of H₂O₂ -generation). rather than changes in redox potential or transportation of the metal-ion. Evidence is also presented for a mechanism in which Fe(II) and H₂O₂ react inside the mitochondrial matrix.

Ascorbate (vitamin C) is shown to be pro-oxidant in this system. except when present at very high concentration when it becomes antioxidant in nature.     

The Control of Iron-Induced Oxidative Damage in Isolated Rat-Liver Mitochondria by Respiration State and Ascorbate

The reaction of iron (II) with H₂O₂ is believed to generate highly reactive species (e.g., OH) capable of initiating biological damage. This study investigates the possibility that the severity of oxidative damage induced by iron in hepatic mitochondria is determined by the level of mitochondrial-H₂O₂ generation, which is believed to be particularly prominent in state-4 respiration.

Iron-induced damage is found to be greater in state-4 than in state-3 respiration. Experiments using uncoupling agents and Ca⁺⁺ to mimic state-3 conditions indicate that this effect reflects differences in the steady-state oxidation-level of the electron carriers of the respiratory chain (and hence the level of H₂O₂ -generation). rather than changes in redox potential or transportation of the metal-ion. Evidence is also presented for a mechanism in which Fe(II) and H₂O₂ react inside the mitochondrial matrix.

Ascorbate (vitamin C) is shown to be pro-oxidant in this system. except when present at very high concentration when it becomes antioxidant in nature.     

Structure-Related Oxidative Damage in Rat Brain After Acute and Chronic Electroshock

The role of oxidative stress in electroconvulsive therapy-related effects is not well studied. The purpose of this study was to determine oxidative stress parameters in several brain structures after a single electroconvulsive seizure or multiple electroconvulsive seizures. Rats were given either a single electroconvulsive shock or a series of eight electroconvulsive shocks. Brain regions were isolated, and levels of oxidative stress in the brain tissue (cortex, hippocampus, striatum and cerebellum) were measured. We demonstrated a decrease in lipid peroxidation and protein carbonyls in the hippocampus, cerebellum, and striatum several times after a single electroconvulsive shock or multiple electroconvulsive shocks. In contrast, lipid peroxidation increases both after a single electroconvulsive shock or multiple electroconvulsive shocks in cortex. In conclusion, we demonstrate an increase in oxidative damage in cortex, in contrast to a reduction of oxidative damage in hippocampus, striatum, and cerebellum.     

Role of Fe(III) in Fe(II)citrate-mediated peroxidation of mitochondrial membrane lipids

In this report we study the effect of Fe(III) on lipid peroxidation induced by Fe(II)citrate in mitochondrial membranes, as assessed by the production of thiobarbituric acid-reactive substances and antimycin A-insensitive oxygen uptake. The presence of Fe(III) stimulates initiation of lipid peroxidation when low citrate:Fe(II) ratios are used ( 4:1). For a citrate:total iron ratio of 1:1 the maximal stimulation of lipid peroxidation by Fe(III) was observed when the Fe(II):Fe(III) ratio was in the range of 1:1 to 1:2. The lag phase that accompanies oxygen uptake was greatly diminished by increasing concentrations of Fe(III) when the citrate:total iron ratio was 1:1, but not when this ratio was higher. It is concluded that the increase of lipid peroxidation by Fe(III) is observed only when low citrate:Fe(II) ratios were used. Similar results were obtained using ATP as a ligand of iron. Monitoring the rate of spontaneous Fe(II) oxidation by measuring oxygen uptake in buffered medium, in the absence of mitochondria, Fe(III)-stimulated oxygen consumption was observed only when a low citrate:Fe(II) ratio was used. This result suggests that Fe(III) may facilitate the initiation and/or propagation of lipid peroxidation by increasing the rate of Fe(II)citrate-generated reactive oxygen species.     

Glycosaminoglycans reduce oxidative damage induced by copper (Cu+2), iron (Fe+2) and hydrogen peroxide (H2O2) in human fibroblast cultures

Acid glycosaminoglycans (GAGs) antioxidant activity was assessed in a fibroblast culture system by evaluating reduction of oxidative system-induced damage. Three different methods to induce oxidative stress in human skin fibroblast cultures were used. In the first protocol cells were treated with CuSO₄ plus ascorbate. In the second experiment fibroblasts were exposed to FeSO₄ plus ascorbate. In the third system H₂O₂ was utilised. The exposition of fibroblasts to each one of the three oxidant systems caused inhibition of cell growth and cell death, increase of lipid peroxidation evaluated by the analysis of malondialdehyde (MDA), decrease of reduced glutathione (GSH) and superoxide dismutase (SOD) levels, and rise of lactate dehydrogenase activity (LDH). The treatment with commercial GAGs at different doses showed beneficial effects in all oxidative models. Hyaluronic acid (HA) and chondroitin-4-sulphate (C4S) exhibited the highest protection. However, the cells exposed to CuSO₄ plus ascorbate and FeSO₄ plus ascorbate were better protected by GAGs compared to those exposed to H₂O₂. These outcomes confirm the antioxidant properties of GAGs and further support the hypothesis that these molecules may function as metal chelators. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of Sclerin on Energy-linked Functions and Phospholipase Activity in Mitochondria Isolated from Rat Liver and Some Plants

Sclerin (SCL) not only elevated the respiratory control ratio and ADP/O ratio in mitochondria isolated from rat liver and some plants, but was effective in maintaining the energy-linked functions in these mitochondria during aging. There was a close relationship in the effect of SCL between the liberation of fatty acid and maintenance of the energy-linked functions in mitochondria during aging. The liberation of fatty acid was mainly due to the digestion of mitochondrial phospholipids by endogenous phospholipase. SCL had no effect on the activity of phospholipase and rather raised the level of endogenous phospholipase in mitochondria during aging at 30°C. The activity of phospholipase in mitochondria was inhibited by ATP, but stimulated by DNP. It was supposed that SCL inhibits the activity of phospholipase through ATP or high-energy intermediates which is maintained in mitochondria during aging. SCL had a protective effect on the activity of DNP-activated ATPase in mitochondria stored in the cold, and, at a very low concentration, stimulated the ATP-driven NAD reduction by mitochondria.     

Aggressive and non-aggressive personalities differ in oxidative status in selected lines of mice (Mus musculus)

Mice selected for aggression and coping (long attack latency (LAL), reactive coping strategy; short attack latency (SAL), pro-active coping strategy) are a useful model for studying the physiological background of animal personalities. These mice also show a differential stress responsiveness, especially in terms of hypothalamic-pituitary-adrenal axis reactivity, to various challenges. Since the stress response can increase the production of reactive oxygen species, we predicted that the basic oxidative status of the lines could differ. We found that LAL showed higher serum antioxidant capacity (OXY) than SAL, while no differences emerged for reactive oxygen metabolites (ROMs) or the balance between ROMs and OXY, reflecting oxidative stress. Moreover, the lines showed inverse relationships between ROMs or OXY and body mass corrected for age. The results indicate that variation in oxidative status is heritable and linked to personality. This suggests that different animal personalities may be accompanied by differences in oxidative status, which may predict differences in longevity.     

The protective effect of melatonin against cypermethrin-induced oxidative stress damage in Spodoptera litura (Lepidoptera: Noctuidae)

Antioxidant enzymes form the first-line defense against free radicals damage in organisms. Their regulation depends mainly on the oxidant and antioxidant status of the cell, given that oxidants are their principal modulators. Therefore, the aim of the present study was to investigate the effect of melatonin on synthetic pyrethroid insecticide-induced antioxidative enzymes activity in Spodoptera litura larvae. In addition, activities of enzymatic antioxidants viz. superoxide dismutase (SOD), glutathione S-transferase (GST), catalase (CAT), glutathione reductase (GR), α, β-esterase, and acetylcholine esterase (AChE) were assessed. There was no significant change in GST levels in the melatonin-treated groups. Melatonin modulates cypermethrin-induced changes in the activities of esterase and AChE, whereas SOD, CAT, and GR activity was significantly increased in melatonin-treated samples when compared to control. In conclusion, the results of the current study revealed that SP toxicity activated oxidant systems in all antioxidant systems in some tissues of insects. Melatonin administration led to a marked increase in antioxidant activity and inhibited GST and AChE in most of the tissues studied.     

Early Histological and Functional Effects of Chronic Copper Exposure in Rat Liver

Cu is an essential trace element capable of producing toxic effects in animals and man when ingested acutely or chronically in excess. Although chronic Cu exposure is increasingly recognized as a public health issue, its early effects remain largely unknown. We approached the significance of a moderate chronic Cu load in young rats to correlate early hepatic histopathological changes with functional alterations of liver cells. For this purpose, supplementation with 1200 ppm of Cu in rat food for 16 weeks was chosen. In these conditions, Cu load elicited a significant decrease in growth curves. There were mild light microscopy alterations in Cu-treated rats, although increasing intracellular Cu storage was correlated with longer Cu exposure both by histological and biochemical measurements. Ultrastructural alterations included lysosomal inclusions as well as mitochondrial and nuclear changes. Liver perfusion studies revealed higher rates of basal O₂ consumption and colloidal carbon-induced O₂ uptake in Cu-treated rats, with enhanced carbon-induced O₂/carbon uptake ratios and NF-κB DNA binding activity. These changes were time-dependent and returned to control values after 12 or16 weeks. It is concluded that subchronic Cu loading in young rats induces early hepatic morphological changes, with enhancement in Küpffer cell-dependent respiratory burst activity and NF-κB DNA binding, cellular responses that may prevent or alleviate the hepatotoxicity of the metal.     

Microcalorimetric Investigation on Metabolic Activity and Effects of La (III) in Mitochondria Isolated from Indica Rice 9311

Thermogenic metabolic curves were determined by the ampoule method at 303 K using a TAM air isothermal microcalorimeter in mitochondria isolated from rice 9311 (Oryza sativa L). From the thermogenic curves the activity recovery rate constant k and the maximum heat power P _m were obtained. Both were positively correlated to the protein content of rice mitochondria. The corresponding correlation coefficients were 0.9959 and 0.9950, respectively, indicating that the in vitro metabolic activity of mitochondria can be reliably expressed by these parameters. Addition of La (III) ions in concentrations ranging from 0 to 130 μg/mL resulted in significantly higher k and P _m values. Concentrations from 140 to 180 μg/mL had the opposite effect. These results are consistent with previous reports on the effects of rare earth elements on plant growth. We propose that the lanthanum-induced change of mitochondrial metabolic activity is a possible mechanism by which La (III) ions influence indica rice 9311 growth.     

Fe (Ii)-Chelates Based on Redox-Active Pyridoxal-Betaines as C-Centered Radicals Causing Single- and Double-Strand Scissions to Dna

The ability of 1-[N-Ethoxycarbonylmethylpridoxylidenium]-2-[2-pyridyljhydrazine bromide code name-[L2-9 = L⁺, X^-]-FE(II) chelate [L2-9-Fe(II)] to induce breaks both in the 43kb linear double-strandphage DNA, and in the 4363 base pair supercoiled pBR322 plasmid DNA is herein described. Neither the free ligand nor FE(II) alone demonstrated any effect on the DNA. The cleaving ability is shown to occur instantaneously under strictly anaerobic conditions, either in the presence or absence of the enzyme catalase. It is also shown to be dose dependent. Thus, atDNA: L2-9-Fe(II) molar ratio of 3.7: 1.0, the linear DNA is randomly cleaved into fragments ranging from 23. Ikb to 4.3kb, whereas at approximately 1:1 molar ratio, the range extends down to 2.5kb fragments. By contrast, at 1:2.7 [plasmid DNA]: chelate-Fe(II). molar ratio, a single-strand nick was observed, and a double strand break was noted at a 1:50 ratio ([plasmid DNA]: chelate-Fe(II). A multi-stage redox cycling involving a carbon-centered (L, X^-)-Fe(III) radical capable of transferring an electron to the DNA to form high unstable [DNA]^- anion-radical is invoked to explain the degradation of the chain macromolecule. Possible modes for regeneration of the chelate-Fe(III) radical both at the cell-free and at the cell levels are proposed.     

Involvement of endogenous nitric oxide in myeloperoxidase mediated benzo(a)pyrene induced polymorphonuclear leukocytes injury

The present study was undertaken to investigate the involvement of nitric oxide in the augmentation of benzo(a)pyrene induced cellular injury in polymorphonuclear leukocytes (PMNs). Polymorphs were isolated from the blood collected from Wistar rats treated with and without benzo(a)pyrene (50mg/kg, i.p.) through cardiac puncture. Catalase, superoxide dismutase (SOD), glutathione-s-transferase (GST), myeloperoxidase (MPO) and nitrite content were estimated in PMNs using standard procedures. Inducible nitric oxide synthase (iNOS) and cytochrome P-4501A1 (CYP1A1) expression in PMNs were also analyzed in presence or absence of nitric oxide synthase (NOS) inhibitors, aminoguanidine (AG, 5mM) and L-N^G nitro L-arginine methyl ester (L-NAME, 1mM). A significant augmentation was observed in the nitrite content, activities of superoxide dismutase, MPO and GST and the expressions of iNOS and CYP1A1, however, catalase activity was attenuated in PMNs of benzo(a)pyrene treated rats as compared with their respective controls. AG and L-NAME resulted in a significant attenuation in nitrite content, MPO activity and iNOS expression; however, no significant alteration was observed in CYP1A1 expression. CYP1A1 inhibitor alpha-naphthoflavone inhibited the expression of iNOS in PMNs of benzo(a)pyrene treated animals significantly. The results obtained thus suggest that CYP1A1 induces iNOS expression leading to the generation of endogenous nitric oxide (NO) that could be responsible for the augmentation of myeloperoxidase-mediated benzo(a)pyrene-induced injury in PMNs.     

Selective and Nonselective Effects of 1-Methyl-4- Phenylpyridinium on Oxygen Consumption in Rat Striatal and Hippocampal Slices

Insights into the etiology and pathophysiology of Parkinson's disease may derive from elucidation of the neurotoxic mechanisms of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite, 1-methyl-4-phenylpyridinium (MPP+). In previous studies, MPP+ provoked oxidation of cytochrome b and K+ leakage into the extracellular space of rat striatal slices. Magnitudes of these time-dependent responses were far greater than expected had the MPP+ effects been limited to dopaminergic terminals. To determine whether cytochromes become oxidized from K(+)-induced increases in ion transport activity or from electron transport inhibition at complex I, oxygen consumption was measured because this should be increased by the former and decreased by the latter mechanism. Low MPP+ concentrations (1 microM) decreased O2 consumption (approximately 40% in 3 h) in striatal slices. This decrease was diminished by mazindol and did not occur in hippocampal slices. High toxin concentrations (100 microM) inhibited oxygen consumption to a greater extent (approximately 60%) in striatal slices; this inhibition was still greater in hippocampal slices. These results support the hypothesis that acute effects of low ("selective") MPP+ concentrations require the presence of dopaminergic terminals to trigger a sequence of destructive metabolic events but that the metabolic consequences of MPP+ spread to neighboring cells. In contrast, high MPP+ concentrations nonselectively inhibit metabolic and ion transport activity without requiring the presence of dopaminergic terminals. These results also suggest that physiological effects of "selective" MPP+ concentrations extend to nondopaminergic cells.     

Malondialdehyde and 4-Hydroxy-2-Nonenal in Plant Tissue Cultures: LC-MS Determination of2, 4-Dinitrophenylhydrazone Derivatives

The cytologically active secondary lipid peroxidation products, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) have been detected as their2, 4-dinitro-phenylhydrazone (DNP) derivatives in plant tissue cultures using LC-MS. This paper reports, for the first time, the use of LC-MS methodology to definitively identify 4-hydroxy-2-nonenal in plants. Limits of detection for the two derivatives are approximately 5pmol (1.2 × 10^-9g; 1μM) and O.1pmol (3 × 10^-l1g; 20nM) respectively. Mass spectrometer response was linear in the range from 2-200μM DNP-MDA and 0.02-10μM DNP-HNE.

This methodology has been used to assess the formation of aldehydic secondary lipid peroxidation products in dedifferentiated callus cultures of Daucus carota. The finding that profiles of MDA and HNE can be correlated with embryogenic competence is of considerable interest as oxidative status has already been implicated as a regulatory factor in animal development.     

Protein-aggregating ability of different protoporphyrin-IX nanostructures is dependent on their oxidation and protein-binding capacity

Porphyrias are rare blood disorders caused by genetic defects in the heme biosynthetic pathway and are associated with the accumulation of high levels of porphyrins that become cytotoxic. Porphyrins, due to their amphipathic nature, spontaneously associate into different nanostructures, but very little is known about the cytotoxic effects of these porphyrin nanostructures. Previously, we demonstrated the unique ability of fluorescent biological porphyrins, including protoporphyrin-IX (PP-IX), to cause organelle-selective protein aggregation, which we posited to be a major mechanism by which fluorescent porphyrins exerts their cytotoxic effect. Herein, we tested the hypothesis that PP-IX-mediated protein aggregation is modulated by different PP-IX nanostructures via a mechanism that depends on their oxidizing potential and protein-binding ability. UV–visible spectrophotometry showed pH-mediated reversible transformations of PP-IX nanostructures. Biochemical analysis showed that PP-IX nanostructure size modulated PP-IX-induced protein oxidation and protein aggregation. Furthermore, albumin, the most abundant serum protein, preferentially binds PP-IX dimers and enhances their oxidizing ability. PP-IX binding quenched albumin intrinsic fluorescence and oxidized His-91 residue to Asn/Asp, likely via a previously described photo-oxidation mechanism for other proteins. Extracellular albumin protected from intracellular porphyrinogenic stress and protein aggregation by acting as a PP-IX sponge. This work highlights the importance of PP-IX nanostructures in the context of porphyrias and offers insights into potential novel therapeutic approaches.     

Circadian Rhythms of Oxidative Phosphorylation: Effects of Rotenone and Melatonin on Isolated Rat Brain Mitochondria

Mitochondrial experiments are of increasing interest in different fields of research. Inhibition of mitochondrian activities seems to play a role in Parkinson's disease and in this regard several animal models have used inhibitors of mitochondrial respiration such as rotenone or MPTP. Most of these experiments were done during the daytime. However, there is no reason for mitochondrial respiration to be constant during the 24h. This study investigated the circadian variation of oxidative phosphorylation in isolated rat brain mitochondria and the administration-time-dependent effect of rotenone and melatonin. The respiratory control ratio, state 3 and state 4, displayed a circadian fluctuation. The highest respiratory control ratio value (3.01) occurred at 04:00h, and the lowest value (2.63) at 08:00h. The highest value of state 3 and state 4 oxidative respiration occurred at 12:00h and the lowest one at 20:00h. The 24h mean decrease in the respiratory control ratio following incubation with melatonin and rotenone was 7 and 32%, respectively; however, the exact amount of the inhibition exerted by these agents varied according to the time of the mitochondria isolation. Our results show the time of mitochondrial isolation could lead to interindividual variability. When studies require mitochondrial isolation from several animals, the time between animal experiments has to be minimized. In oxidative phosphorylation studies, the time of mitochondria isolation must be taken into account, or at least specified in the methods section.     

Oxidative-stress-induced production of pyocyanin by Xanthomonas campestris and its effect on the indicator target organism, Escherichia coli

Pyocyanin, a potential antimicrobial agent, was secreted by Xanthomonas campestris. Treatments with agents causing oxidative stress in the organism caused up to 4.4-fold increase in pyocyanin production. Pyocyanin added in the extracellular space did not affect growth rate of X. campestris, but decreased maximum cell concentration and specific product formation. However, the growth of Escherichia coli, the indicator target organism, was affected by pyocyanin. There was also a significant increase in the intracellular reactive oxygen species (ROS) concentration and antioxidant enzyme [catalase, superoxide dismutase (SOD)] concentrations, in the presence of pyocyanin. The intracellular ROS concentrations in E. coli formed upon exposure to pyocyanin, which is an indicator of the toxicity, was dependent on the growth phase of the organism. Studies with mutants of E. coli showed that intracellular ROS concentration was not significantly affected by the absence of the regulon OxyR, but, was significantly higher in cases when the regulon rpoS or the genes katG or katE were absent. Journal of Industrial Microbiology & Biotechnology (2000) 25, 266–272. Received 08 May 2000/ Accepted in revised form 04 August 2000     

Effects of 1-Methyl-4-Phenylpyridinium on Isolated Rat Brain Mitochondria: Evidence for a Primary Involvement of Energy Depletion

Abstract: The effects of 1-methyl-4-phenylpyridinium (MPP⁺) on the oxygen consumption, ATP production, H₂O₂ production, and mitochondrial NADH-CoQ₁ reductase (complex I) activity of isolated rat brain mitochondria were investigated. Using glutamate and malate as substrates, concentrations of 10–100 µ M MPP⁺ had no effect on state 4 (−ADP) respiration but decreased state 3 (+ADP) respiration and ATP production. Incubating mitochondria with ADP for 30 min after loading with varying concentrations of MPP⁺ produced a concentration-dependent decrease in H₂O₂ production. Incubation of mitochondria with ADP for 60 min after loading with 100 µ M MPP⁺ caused no loss of complex I activity after washing of MPP⁺ from the mitochondrial membranes. These data are consistent with MPP⁺ initially binding specifically to complex I and inhibiting both the flow of reducing equivalents and the production of H₂O₂ by the mitochondrial respiratory chain, without irreversibly damaging complex I. However, mitochondria incubated with H₂O₂ in the presence of Cu²⁺ ions showed decreased complex I activity. This study provides additional evidence that cellular damage initiated by MPP⁺ is due primarily to energy depletion caused by specific binding to complex I, any increased damage due to free radical production by mitochondria being a secondary effect.     

Effect of Chronic Variate Stress on Thiobarbituric-Acid Reactive Species and on Total Radical-Trapping Potential in Distinct Regions of Rat Brain

It has been suggested that oxidative stress is involved in aging and neuropathologic disorders. In addition, chronic stress and high corticosterone levels are suggested to induce neuronal death. The aim of this study is to verify the effect of chronic variate stress on lipoperoxidation and on the total radical-trapping potential (TRAP) in hippocampus, hypothalamus and cerebral cortex. Adult male Wistar rats were submitted to different stressors during 40 days. Lipid peroxide levels were assessed by the thiobarbituric acid reactive species (TBARS) reaction, and TRAP was measured by the decrease in luminescence using the 2-2-azo-bis(2-amidinopropane)-luminol system. The results showed that in cerebral cortex homogenates chronic stress induces an increase in oxidative stress. In hypothalamus a decreased lipoperoxidation was observed, however TRAP showed no difference. In hippocampus no difference was observed. We concluded that prolonged stress induces oxidative stress which varies selectively with the brain region.