Measuring "free" iron levels in Caenorhabditis elegans using low-temperature Fe(III) electron paramagnetic resonance spectroscopy

Oxidative stress, caused by free radicals within the body, has been associated with the process of aging and many human diseases. Because free radicals, in particular superoxide, are difficult to measure, an alternative indirect method for measuring oxidative stress levels has been used successfully in Escherichia coli and yeast. This method is based on a proposed connection between elevated superoxide levels and release of iron from solvent-exposed [4Fe-4S] enzyme clusters that eventually leads to an increase in hydroxyl radical production. In past studies using bacteria and yeast, a positive correlation was found between superoxide production or oxidative stress due to superoxide within the organism and electron paramagnetic resonance (EPR) detectable "free" iron levels. In the current study, we have developed a reliable and efficient method for measuring "free" iron levels in Caenorhabditis elegans using low-temperature Fe(III) EPR at g=4.3. This method uses synchronized worm cultures grown on plates that are homogenized and treated with desferrioxamine, an Fe(III) chelator, prior to packing the EPR tube. Homogenization was found not to alter "free" iron levels, whereas desferrioxamine treatment significantly raised these levels, indicating the presence of both Fe(II) and Fe(III) in the "free" iron pool. The correlation between free radical levels and the observed "free" iron levels was examined by using heat stress and paraquat treatment. The intensity of the Fe(III) EPR signal, and thus the concentration of the "free" iron pool, varied with the treatments that altered radical levels without changing the total iron levels. This study provides the groundwork needed to uncover the correlation among oxidative stress, "free" iron levels, and longevity in C. elegans.     

Evidence for a novel role of copper-zinc superoxide dismutase in zinc metabolism

The LYS7 gene in Saccharomyces cerevisiae encodes a protein (yCCS) that delivers copper to the active site of copper-zinc superoxide dismutase (CuZn-SOD, a product of the SOD1 gene). In yeast lacking Lys7 (lys7Delta), the SOD1 polypeptide is present but inactive. Mutants lacking the SOD1 polypeptide (sod1Delta) and lys7Delta yeast show very similar phenotypes, namely poor growth in air and aerobic auxotrophies for lysine and methionine. Here, we demonstrate certain phenotypic differences between these strains: 1) lys7Delta cells are slightly less sensitive to paraquat than sod1Delta cells, 2) EPR-detectable or "free" iron is dramatically elevated in sod1Delta mutants but not in lys7Delta yeast, and 3) although sod1Delta mutants show increased sensitivity to extracellular zinc, the lys7Delta strain is as resistant as wild type. To restore the SOD catalytic activity but not the zinc-binding capability of the SOD1 polypeptide, we overexpressed Mn-SOD from Bacillus stearothermophilus in the cytoplasm of sod1Delta yeast. Paraquat resistance was restored to wild-type levels, but zinc was not. Conversely, expression of a mutant CuZn-SOD that binds zinc but has no SOD activity (H46C) restored zinc resistance but not paraquat resistance. Taken together, these results strongly suggest that CuZn-SOD, in addition to its antioxidant properties, plays a role in zinc homeostasis.     

Unincorporated iron pool is linked to oxidative stress and iron levels in Caenorhabditis elegans

Free radicals or reactive oxygen species (ROS) are relatively short-lived and are difficult to measure directly; so indirect methods have been explored for measuring these transient species. One technique that has been developed using Escherichia coli and Saccharomyces cerevisiae systems, relies on a connection between elevated superoxide levels and the build-up of a high-spin form of iron (Fe(III)) that is detectable by electron paramagnetic resonance (EPR) spectroscopy at g?=?4.3. This form of iron is referred to as "free" iron. EPR signals at g?=?4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S?=?5/2) Fe(III) ions in sites of low symmetry. Unincorporated iron in this study refers to this high-spin Fe(III) that is captured by desferrioxamine which is detected by EPR at g value of 4.3. Previously, we published an adaptation of Fe(III) EPR methodology that was developed for Caenorhabditis elegans, a multi-cellular organism. In the current study, we have systematically characterized various factors that modulate this unincorporated iron pool. Our results demonstrate that the unincorporated iron as monitored by Fe(III) EPR at g?=?4.3 increased under conditions that were known to elevate steady-state ROS levels in vivo, including: paraquat treatment, hydrogen peroxide exposure, heat shock treatment, or exposure to higher growth temperature. Besides the exogenous inducers of oxidative stress, physiological aging, which is associated with elevated ROS and ROS-mediated macromolecular damage, also caused a build-up of this iron. In addition, increased iron availability increased the unincorporated iron pool as well as generalized oxidative stress. Overall, unincorporated iron increased under conditions of oxidative stress with no change in total iron levels. However, when total iron levels increased in vivo, an increase in both the pool of unincorporated iron and oxidative stress was observed suggesting that the status of the unincorporated iron pool is linked to oxidative stress and iron levels.     

Antioxidant activity of L-ascorbic acid in wild-type and superoxide dismutase deficient strains of Saccharomyces cerevisiae

Much has been published on the non-enzymatic antioxidant L-ascorbic acid (vitamin C), but even so its interaction with endogenous cellular defense systems has not yet been fully elucidated. Our study investigated the antioxidant activity of L-ascorbic acid in wild-type strain EG103 (SOD) Saccharomyces cerevisiae and isogenic mutant strains deficient in cytosolic superoxide dismutase (sod1delta), mitochondrial superoxide dismutase (sod2delta) or both (sod1delta sod2delta), metabolizing aerobically or anaerobically with and without the stressing agent paraquat. The results show that during both aerobic and anaerobic metabolism there was a significant increase in the survival of both wild-type S. cerevisiae cells and the mutant cells (sod1delta, sod2delta and sod1delta sod2delta) when pretreated with L-ascorbic acid before exposure to paraquat. Exposure to paraquat resulted in higher catalase activity but this significantly decreased when the cells were pre-treated with L-ascorbic acid. These results demonstrate that due to the damage caused by paraquat, the antioxidant protection of L-ascorbic acid seems to be mediated by catalase levels in yeast cells.     

Mutations in Saccharomyces cerevisiae iron-sulfur cluster assembly genes and oxidative stress relevant to Cu,Zn superoxide dismutase

Saccharomyces cerevisiae lacking Cu,Zn superoxide dismutase (SOD1) show several metabolic defects including aerobic blockages in methionine and lysine biosynthesis. We have previously shown that mutations in genes implicated in the formation of iron-sulfur clusters, designated seo (suppressors of endogenous oxidation), reverse the oxygen-dependent methionine and lysine auxotrophies of a sod1Delta strain. We now report the surprising finding that seo mutants do not reduce oxidative damage as shown by the lack of reduction of EPR-detectable "free" iron, which is characteristic of sod1Delta mutants. In fact, they exhibit increased oxidative damage as evidenced by increased accumulation of protein carbonyls. The seo class of mutants overaccumulates mitochondrial iron, and this iron accumulation is critical for suppression of the sod1Delta biosynthetic defects. Blocking overaccumulation of mitochondrial iron abolished the ability of the seo mutants to suppress the sod1Delta auxotrophies. By contrast, increasing the mitochondrial iron content of sod1Delta yeast using high copy MMT1, which encodes a mitochondrial iron transporter, was sufficient to mimic the seo mutants. Our studies indicated that suppression of the sod1Delta methionine auxotrophy was dependent on the pentose phosphate pathway, which is a major source of NADPH production. By comparison, the sod1Delta lysine auxotrophy appears to be reversed in the seo mutants by increased expression of genes in the lysine biosynthetic pathway, perhaps through sensing of mitochondrial damage by the retrograde response.     

Protective role of mitochondrial superoxide dismutase against high osmolarity,heat and metalloid stress in<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Superoxide dismutases, both cytosolic Cu, Zn-SOD encoded by SOD1 and mitochondrial Mn-SOD encoded by SOD2, serve Saccharomyces cerevisiae cells for defense against the superoxide radical but the phenotypes of sod1A and sod2delta mutant strains are different. Compared with the parent strain and the sod1delta mutant, the sod2delta mutant shows a much more severe growth defect at elevated salt concentrations, which is partially rescued by 2 mmol/L glutathione. The growth of all three strains is reduced at 37 degrees C, the sod2delta showing the highest sensitivity, especially when cultured in air. Addition of 1 mmol/L glutathione to the medium restores aerobic growth of the sod1delta mutant but has only a minor effect on the growth of the sod2delta strain at 37 degrees C. The sod2delta strain is also sensitive to AsIIl and AsV and its sensitivity is much more pronounced under aerobic conditions. These results suggest that, unlike the Sodlp protein, whose major role is oxidative stress defense, Sod2p also plays a role in protecting S. cerevisiae cells against other stresses--high osmolarity, heat and metalloid stress.     

Protective role of superoxide dismutases against ionizing radiation in yeast

The protective role of superoxide dismutases (SODs) against ionizing radiation, which generates reactive oxygen species (ROS) harmful to cellular function, was investigated in the wild-type and in mutant yeast strains lacking cytosolic CuZnSOD (sod1Delta), mitochondrial MnSOD (sod2Delta), or both SODs (sod1Deltasod2Delta). Upon exposure to ionizing radiation, there was a distinct difference between these strains in regard to viability and the level of protein carbonyl content, which is the indicative marker of oxidative damage to protein, intracellular H2O2 level, as well as lipid peroxidation. When the oxidation of 2',7'-dichlorofluorescin was used to examine the hydroperoxide production in yeast cells, the SOD mutants showed a higher degree of increase in fluorescence upon exposure to ionizing radiation as compared to wild-type cells. These results indicated that mutants deleted for SOD genes were more sensitive to ionizing radiation than isogenic wild-type cells. Induction and inactivation of other antioxidant enzymes, such as catalase, glucose 6-phosphate dehydrogenase, and glutathione reductase, were observed after their exposure to ionizing radiation both in wild-type and in mutant cells. However, wild-type cells maintained significantly higher activities of antioxidant enzymes than did mutant cells. These results suggest that both CuZnSOD and MnSOD may play a central role in protecting cells against ionizing radiation through the removal of ROS, as well as in the protection of antioxidant enzymes.     

Interaction between 6-hydroxydopamine and transferrin: "Let my iron go"

The dopamine analogue 6-hydroxydopamine (6-OHDA) is selectively toxic to catecholaminergic neurons. Because of its selectivity for neuroblastic cells in the sympathetic nervous system lineage, 6-OHDA has been suggested as a chemotherapeutic agent for targeted treatment of patients with neuroblastoma. We tested the hypothesis that the toxicity of 6-OHDA is caused by its interaction with serum ferric transferrin (Fe-TF) resulting in release of iron. We further hypothesized that this iron, through its redox-cycling by 6-OHDA, triggers generation of reactive oxygen species. 6-OHDA-induced release of iron from Fe-TF was demonstrated by: (1) low-temperature EPR spectroscopic evidence for decay of the characteristic Fe-TF signal (g = 4.3) and appearance of the high-spin signal from iron chelated by 6-OHDA oxidation products; (2) spectrophotometric detection of complexing of iron with the Fe(2+) chelator ferrozine; (3) redox-cycling of ascorbate yielding EPR-detectable ascorbate radicals; and (4) generation of hydroxyl radicals as evidenced by EPR spectroscopy of their adduct with a spin trap, 5, 5'-dimethylpyrroline oxide (DMPO) (DMPO-OH). Our low-temperature EPR studies showed that in human plasma, 6-OHDA caused iron release only under nitrogen gas but not under air or oxygen. The absence of a 6-OHDA effect in plasma under aerobic conditions was most likely due to its ferroxidase activity [with consequent reuptake of Fe(III) by apoTF] and catalytic oxidation of 6-OHDA by ceruloplasmin. Modeling of these plasma activities by a stable nitroxide radical, 2,2,6, 6-tetramethyl-1-piperidinyloxy (TEMPOL), resulted in protection of plasma Fe-TF against iron release under nitrogen. Parenteral administration of 6-OHDA to mice resulted in iron release from Fe-TF as evidenced by transformation of the Fe-TF low-temperature EPR signal that was indistinguishable from that seen in in vitro models. In addition, administration of the iron chelator deferoxamine (DFO) to mice prior to administration of toxic doses of 6-OHDA resulted in a decrease in activity impairment of mice as compared to that seen with 6-OHDA alone. These findings underscore the physiological and pharmacological relevance of 6-OHDA-mediated iron release from Fe-TF and suggest that iron chelators (DFO) may be used for prevention of 6-OHDA toxicity.     

Superoxide inhibits 4Fe-4S cluster enzymes involved in amino acid biosynthesis. Cross-compartment protection by CuZn-superoxide dismutase

Among the phenotypes of Saccharomyces cerevisiae mutants lacking CuZn-superoxide dismutase (Sod1p) is an aerobic lysine auxotrophy; in the current work we show an additional leaky auxotrophy for leucine. The lysine and leucine biosynthetic pathways each contain a 4Fe-4S cluster enzyme homologous to aconitase and likely to be superoxide-sensitive, homoaconitase (Lys4p) and isopropylmalate dehydratase (Leu1p), respectively. We present evidence that direct aerobic inactivation of these enzymes in sod1 Delta yeast results in the auxotrophies. Located in the cytosol and intermembrane space of the mitochondria, Sod1p likely provides direct protection of the cytosolic enzyme Leu1p. Surprisingly, Lys4p does not share a compartment with Sod1p but is located in the mitochondrial matrix. The activity of a second matrix protein, the tricarboxylic acid cycle enzyme aconitase, was similarly lowered in sod1 Delta mutants. We measured only slight changes in total mitochondrial iron and found no detectable difference in mitochondrial "free" (EPR-detectable) iron making it unlikely that a gross defect in mitochondrial iron metabolism is the cause of the decreased enzyme activities. Thus, we conclude that when Sod1p is absent a lysine auxotrophy is induced because Lys4p is inactivated in the matrix by superoxide that originates in the intermembrane space and diffuses across the inner membrane.     

Adaptative response to enhanced basal oxidative damage in sod mutants from Saccharomyces cerevisiae

We investigated the adaptative response of S. cerevisiae in sod mutants (sod1Δ, sod2Δ and sod1Δsod2Δ) after H₂O₂ treatment in the stationary phase. sod2Δ and sod1Δsod2Δ demonstrated the highest levels of GSH in the control, suggesting that pathways which include GSH protect these double mutants against oxidative stress. In addition, sod1Δ and sod1Δsod2Δ had higher iron levels than the wild-type, independently of H₂O₂ stress. Fe levels were increased in sod2Δ following H₂O₂ In addition, the sod2Δ mutant was more sensitive to H₂O₂ treatment than the wild-type. These results suggest that sod2Δ sensibility may be associated with •OH production by the Fenton reaction. This increased iron demand in the sod2Δ mutant may be a reflection of the cells’ efforts to reconstitute proteins that are inactivated in conditions of excess superoxide. MDA levels were assayed by HPLC in these mutants. The highest MDA levels could be observed after 10mM H₂O₂ treatment in the sod1Δsod2Δ double mutant. After treatment with a GSH inhibitor, the MDA level was still higher in the same strain. Thus, both direct and indirect GSH pathways are involved in the protection of lipid membranes and proteins in these mutants and may constitute an adaptative response to enhanced basal oxidative damage produced by superoxide.     

Yeast frataxin mutants display decreased superoxide dismutase activity crucial to promote protein oxidative damage

Iron overload is involved in several pathological conditions, including Friedreich ataxia, a disease caused by decreased expression of the mitochondrial protein frataxin. In a previous study, we identified 14 proteins selectively oxidized in yeast cells lacking Yfh1, the yeast frataxin homolog. Most of these were magnesium-binding proteins. Decreased Mn-SOD activity, oxidative damage to CuZn-SOD, and increased levels of chelatable iron were also observed in this model. This study explores the relationship between low SOD activity, the presence of chelatable iron, and protein damage. We observed that addition of copper and manganese to the culture medium restored SOD activity and prevented both oxidative damage and inactivation of magnesium-binding proteins. This protection was compartment specific: recovery of mitochondrial enzymes required the addition of manganese, whereas cytosolic enzymes were recovered by adding copper. Copper treatment also decreased Δyfh1 sensitivity to menadione. Finally, a Δsod1 mutant showed high levels of chelatable iron and inactivation of magnesium-binding enzymes. These results suggest that reduced superoxide dismutase activity contributes to the toxic effects of iron overloading. This would also apply to pathologies involving iron accumulation.     

Function of oxygen resistance proteins in the anaerobic,sulfate-reducing bacterium Desulfovibrio vulgaris hildenborough

Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H(2)O(2)) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase. The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H(2)O(2) resistance may also be conferred by two other rbr genes in the D. vulgaris genome. Inhibition of Sod activity by azide and H(2)O(2), but not by cyanide, indicated it to be an iron-containing Sod. The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE). A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of D. vulgaris. Thus, Sor plays a key role in oxygen defense of D. vulgaris under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm. Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets.     

Studies on human lactoferrin by electron paramagnetic resonance, fluorescence, and resonance Raman spectroscopy

Investigations of metal-substituted human lactoferrins by fluorescence, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy confirm the close similarity between lactoferrin and serum transferrin. As in the case of Fe(III)- and Cu(II)-transferrin, a significant quenching of apolactoferrin's intrinsic fluorescence is caused by the interaction of Fe(III), Cu(II), Cr(III), Mn(III), and Co(III) with specific metal binding sites. Laser excitation of these same metal-lactoferrins produces resonance Raman spectral features at ca. 1605, 1505, 1275, and 1175 cm-1. These bands are characteristic of tyrosinate coordination to the metal ions as has been observed previously for serum transferins and permit the principal absorption band (lambda max between 400 and 465 nm) in each of the metal-lactoferrins to be assigned to charge transfer between the metal ion and tyrosinate ligands. Furthermore, as in serum transferrin the two metal binding sites in lactoferrin can be distinguished by EPR spectroscopy, particularly with the Cr(III)-substituted protein. Only one of the two sites in lactoferrin allows displacement of Cr(III) by Fe(III). Lactoferrin is known to differ from serum transferrin in its enhanced affinity for iron. This is supported by kinetic studies which show that the rate of uptake of Fe(III) from Fe(III)--citrate is 10 times faster for apolactoferrin than for apotransferrin. Furthermore, the more pronounced conformational change which occurs upon metal binding to lactoferrin is corroborated by the production of additional EPR-detectable Cu(II) binding sites in Mn(III)-lactoferrin. The lower pH required for iron removal from lactoferrin causes some permanent change in the protein as judged by altered rates of Fe(III) uptake and altered EPR spectra in the presence of Cu(II). Thus, the common method of producing apolactoferrin by extensive dialysis against citric acid (pH 2) appears to have an adverse effect on the protein.     

Oxidative stress and iron are implicated in fragmenting vacuoles of Saccharomyces cerevisiae lacking Cu,Zn-superoxide dismutase.

The absence of the antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) is shown here to cause vacuolar fragmentation in Saccharomyces cerevisiae. Wild-type yeast have 1-3 large vacuoles whereas the sod1Delta yeast have as many as 50 smaller vacuoles. Evidence that this fragmentation is oxygen-mediated includes the findings that aerobically (but not anaerobically) grown sod1Delta yeast exhibit aberrant vacuoles and genetic suppressors of other oxygen-dependent sod1 null phenotypes rescue the vacuole defect. Surprisingly, iron also is implicated in the fragmentation process as iron addition exacerbates the sod1Delta vacuole defect while iron starvation ameliorates it. Because the vacuole is reported to be a site of iron storage and iron reacts avidly with reactive oxygen species to generate toxic side products, we propose that vacuole damage in sod1Delta cells arises from an elevation of iron-mediated oxidation within the vacuole or from elevated pools of "free" iron that may bind nonproductively to vacuolar ligands. Furthermore, additional pleiotropic phenotypes of sod1Delta cells (including increased sensitivity to pH, nutrient deprivation, and metals) may be secondary to vacuolar compromise. Our findings support the hypothesis that oxidative stress alters cellular iron homeostasis which in turn increases oxidative damage. Thus, our findings may have medical relevance as both oxidative stress and alterations in iron homeostasis have been implicated in diverse human disease processes. Our findings suggest that strategies to decrease intracellular iron may significantly reduce oxidatively induced cellular damage.     

Mechanisms underlying the cytotoxic effects of Tachpyr--a novel metal chelator

Tachpyr (N,N'N"-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane), a novel metal chelator, was previously shown to deplete intracellular iron and exert a cytotoxic effect on cultured bladder cancer cells. Tachpyr binds Fe(II) and readily reduces Fe(III). The iron(II)-Tachpyr chelate undergoes intramolecular oxidative dehydrogenation resulting in mono- and diimino Fe(II) complexes. The present study investigates the redox-activity of the Tachpyr-iron complex to better define the mechanism of Tachpyr's cytotoxicity. Tachpyr's mechanism of cytotoxicity was studied using cell-free solutions, isolated DNA, and cultured mammalian cells by employing UV-VIS spectrophotometry, oximetry, spin-trapping technique, and electron paramagnetic resonance (EPR) spectrometry. The results show that: (1) Tachpyr by itself after 24 h of incubation had a cytotoxic effect on cultured cells; (2) fully oxidized Tachpyr had no cytotoxic effects on cultured cells even after 24 h of incubation; (3) Tachpyr protected isolated DNA against H(2)O(2)-induced damage, but not against HX/XO-induced damage; and (4) Tachpyr-Fe(II) chelate slows down but does not block oxidation of Fe(II), allows O*(-)(2)-induced or Tachpyr-induced reduction of Fe(III), and consequently promotes production of *OH through the Haber-Weiss reaction cycle. The results indicate that Tachpyr can protect cells against short-term, metal-mediated damage. However, upon prolonged incubation, Tachpyr exerts cytotoxic effects. Therefore, in addition to iron depletion, low-level oxidative stress, which in part occurs because of redox cycling of the coordinated iron ion, may contribute to the cytotoxic effects of Tachpyr.     

A new function of the Desulfovibrio vulgaris Hildenborough [Fe] hydrogenase in the protection against oxidative stress

Sulfate-reducing bacteria, like Desulfovibrio vulgaris Hildenborough, have developed a set of reactions allowing them to survive in oxic environments and even to reduce molecular oxygen to water. D. vulgaris contains a cytoplasmic superoxide reductase (SOR) and a periplasmic superoxide dismutase (SOD) involved in the elimination of superoxide anions. To assign the function of SOD, the periplasmic [Fe] hydrogenase activity was followed in both wild-type and sod deletant strains. This activity was lower in the strain lacking the SOD than in the wild-type when the cells were exposed to oxygen for a short time. The periplasmic SOD is thus involved in the protection of sensitive iron-sulfur-containing enzyme against superoxide-induced damages. Surprisingly, production of the periplasmic [Fe] hydrogenase was higher in the cells exposed to oxygen than in those kept in anaerobic conditions. A similar increase in the amount of [Fe] hydrogenase was observed when an increase in the redox potential was induced by addition of chromate. Viability of the strain lacking the gene encoding [Fe] hydrogenase after exposure to oxygen for 1 h was lower than that of the wild-type. These data reveal for the first time that production of the periplasmic [Fe] hydrogenase is up-regulated in response to an oxidative stress. A new function of the periplasmic [Fe] hydrogenase in the protective mechanisms of D. vulgaris Hildenborough toward an oxidative stress is proposed.     

Inhibition of phagocytic activity of ARPE-19 cells by free radical mediated oxidative stress

Oxidative stress is a main factor responsible for key changes leading to the onset of age-related macular degeneration (ARMD) that occur in the retinal pigment epithelium (RPE), which is involved in phagocytosis of photoreceptor outer segments (POS). In this study, hydrogen peroxide (H₂O₂), H₂O₂ and iron ions (Fe) or rose Bengal (RB) in the presence of NADH and Fe were used to model free radical mediated oxidative stress to test if free radicals and singlet oxygen have different efficiency to inhibit phagocytosis of ARPE-19 cells. Free radical mediated oxidative stress was confirmed by HPLC-EC(Hg) measurements of cholesterol hydroperoxides in treated cells. Electron paramagnetic resonance (EPR) spin trapping was employed to detect superoxide anion. Cell survival was analyzed by the MTT assay. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS and non-specific phagocytosis of fluorescent beads were measured by flow cytometry. HPLC analysis of cells photosensitized with RB in the presence of NADH and Fe indicated substantial increase in formation of free radical-dependent 7α/7β-hydroperoxides. EPR spin trapping confirmed the photogeneration of superoxide anion in samples enriched with RB, NADH and Fe. For all three protocols sub-lethal oxidative stress induced significant inhibition of the specific phagocytosis of POS. In contrast, non-specific phagocytosis was inhibited only by H₂O₂ or H₂O₂ and Fe treatment. Inhibition of phagocytosis was transient and recoverable by 24?h. These results suggest that free radicals may exert similar to singlet oxygen efficiency in inhibiting phagocytosis of RPE cells, and that the effect depends on the location where initial reactive species are formed.     

Mitochondrial superoxide decreases yeast survival in stationary phase

Yeast lacking mitochondrial superoxide dismutase (MnSOD) display shortened stationary-phase survival and provide a good model system for studying mitochondrial oxidative damage. We observed a marked decrease in respiratory function preceding stationary-phase death of yeast lacking MnSOD (sod2Delta). Agents (mitochondrial inhibitors) that are known to increase or decrease superoxide production in submitochondrial particles affected stationary-phase survival in a manner inversely correlated with their effects on superoxide production, implicating superoxide in this mitochondrial disfunction. Similar but less-dramatic effects were observed in wild-type yeast. The activities of certain mitochondrial enzymes were particularly affected. In sod2Delta yeast the activity of aconitase, a 4Fe-4S-cluster-containing enzyme located in the matrix, was greatly and progressively decreased as the cells established stationary phase. Succinate dehydrogenase activity also decreased in MnSOD mutants; cytochrome oxidase and ATPase activities did not. Aconitase could be reactivated by addition of materials required for cluster assembly (Fe3+ and a sulfur source), both in extracts and in vivo, indicating that inactivation of the enzyme was by disassembly of the cluster. Our results support the conclusion that superoxide is generated in the mitochondria in vivo and under physiological conditions and that MnSOD is the primary defense against this toxicity. When the balance between superoxide generation and MnSOD activity is disrupted, superoxide mediates iron release from mitochondrial iron-sulfur clusters, leading first to loss of mitochondrial function and then to death, independently of mtDNA damage. These results raise the possibility that similar processes may occur in higher eukaryotes.