DNA damage in rat lymphocytes treated in vitro with iron cations and exposed to 7 mT magnetic fields (static or 50 Hz)

The present study was undertaken to verify a hypothesis that exposure of the cells to static or 50 Hz magnetic fields (MF) and simultaneous treatment with a known oxidant, ferrous chloride, may affect the oxidative deterioration of DNA molecules.The comet assay was chosen for the assessment of DNA damage. The experiments were performed on isolated rat lymphocytes incubated for 3h in Helmholtz coils at 7 mT static or 50 Hz MF. During MF exposure, part of the cell samples were incubated with 0.01 microM H(2)O(2) and another one with 10 microg/ml FeCl(2,) the rest serving as controls.Lymphocyte exposure to MF at 7 mT did not increase the number of cells with DNA damage in the comet assay. Incubation of lymphocytes with 10 microg/ml FeCl(2) did not produce a detectable damage of DNA either. However, when the FeCl(2)-incubated lymphocytes were simultaneously exposed to 7 mT MF, the number of damaged cells was significantly increased and reached about 20% for static MF and 15% for power frequency MF. In the control samples about 97% of the cells did not have any DNA damage.It is not possible at present to offer a reasonable explanation for the findings of this investigation - the high increase in the number of lymphocytes showing symptoms of DNA damage in the comet assay, following simultaneous exposure to the combination of two non-cytotoxic factors -10 microg/ml FeCl(2) and 7 mT MF. In view of the obtained results we can only hypothesise that under the influence of simultaneous exposure to FeCl(2) and static or 50 Hz MF, the number of reactive oxygen species generated by iron cations may increase substantially. Further studies will be necessary to confirm this hypothesis and define the biological significance of the observed effect.     

Dissimilatory iron reduction and odor indicator abatement by biofilm communities in swine manure microcosms

Animal waste odors arising from products of anaerobic microbial metabolism create community relations problems for livestock producers. We investigated a novel approach to swine waste odor reduction: the addition of FeCl(3), a commonly used coagulant in municipal wastewater treatment, to stimulate degradation of odorous compounds by dissimilatory iron-reducing bacteria (DIRB). Two hypotheses were tested: (i) FeCl(3) is an effective source of redox-active ferric iron (Fe(3+)) for dissimilatory reduction by bacteria indigenous to swine manure, and (ii) dissimilatory iron reduction results in significant degradation of odorous compounds within 7 days. Our results demonstrated that Fe(3+) from FeCl(3) was reduced biologically as well as chemically in laboratory microcosms prepared with prefiltered swine manure slurry and limestone gravel, which provided pH buffering and a substrate for microbial biofilm development. Addition of a 1-g liter(-1) equivalent concentration of Fe(3+) from FeCl(3), but not from presynthesized ferrihydrite, caused initial, rapid solids flocculation, chemical Fe(3+) reduction, and E(h) increase, followed by a 2-day lag period. Between 2 and 6 days of incubation, increases in Fe(2+) concentrations were accompanied by significant reductions in concentrations of volatile fatty acids used as odor indicators. Increases in Fe(2+) concentrations between 2 and 6 days did not occur in FeCl(3)-treated microcosms that were sterilized by gamma irradiation or amended with NaN(3), a respiratory inhibitor. DNA sequences obtained from rRNA gene amplicons of bacterial communities in FeCl(3)-treated microcosms were closely related to Desulfitobacterium spp., which are known representatives of DIRB. Use of iron respiration to abate wastewater odors warrants further investigation.     

Oxygen-dependent inactivation of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts of Rhodospirillum rubrum and establishment of a model inactivation system with purified enzyme.

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPC/O) was inactivated in crude extracts of Rhodospirillum rubrum under atmospheric levels of oxygen; no inactivation occurred under an atmosphere of argon. RuBP carboxylase activity did not decrease in dialyzed extracts, indicating that a dialyzable factor was required for inactivation. The inactivation was inhibited by catalase. Purified RuBPC/O is relatively oxygen stable, as no loss of activity was observed after 4 h under an oxygen atmosphere. The aerobic inactivation catalyzed by endogenous factors in crude extracts was mimicked by using a model system containing purified enzyme, ascorbate, and FeSO4 or FeCl3. Dithiothreitol was found to substitute for ascorbate in the model system. Preincubation of the purified enzyme with RuBP led to enhanced inactivation, whereas Mg2+ and HCO3- significantly protected against inactivation. Unlike the inactivation catalyzed by endogenous factors from extracts of R. rubrum, inactivation in the model system was not inhibited by catalase. It is proposed that ascorbate and iron, in the presence of oxygen, generate a reactive oxygen species which reacts with a residue at the activation site, rendering the enzyme inactive.     

Prooxidant action of xanthurenic acid and quinoline compounds: Role of transition metals in the generation of reactive oxygen species and enhanced formation of 8-hydroxy-2′-deoxyguanosine in DNA†

Xanthurenic acid, a product of tryptophan–NAD pathway, and quinoline compounds produced reactive oxygen species as a complex with iron. Aconitase, the most sensitive enzyme to oxidative stress was inactivated effectively by xanthurenic acid and to a lesser extent by 8-quinolinol in the presence of ferrous sulfate. The inactivation of aconitase was iron-dependent, and was prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that reduced iron bound to xanthurenic acid or 8-quinolinol can activate oxygen molecule to form superoxide radical. However, kynurenic acid and quinaldic acid without 8-hydroxyl group did not produce reactive oxygen species. Of the quinoline compounds tested, xanthurenic acid and 8-quinolinol with 8-hydroxyl group stimulated the autooxidation of ferrous ion, but kynurenic acid and quinaldic acid did not affect the oxidation of ferrous ion. Hydroxyl group at 8-positions of quinoline compounds was essential for the binding of iron causing the generation of reactive oxygen species. 8-Quinolinol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2′-deoxyguanosine in DNA, suggesting the quinolinol/copper-dependent stimulation hydroxyl radical formation. Xanthurenic acid and 8-quinolinol as the metal–chelate complexes can show various cytotoxic effects by generating reactive oxygen species through the ferrous or cuprous ion-dependent activation of oxygen molecule. † This paper is dedicated to centennial of the birthday of the late Professor Emeritus Yahito Kotake, a pioneer of the xanthurenic acid research.     

Neuroprotective activity of p-terphenyl leucomentins from the mushroom Paxillus panuoides

The neuroprotective mechanism of p-terphenyl leucomentins from the mushroom Paxillus panuoides was studied. Leucomentins showed potent inhibition of lipid peroxidation and H2O2 neurotoxicity, but free from any role as reactive oxygen species (ROS) scavengers. Iron-mediated oxidative damage has been implicated in these processes, as a provider of ROS via iron. Leucomentins can chelate iron when DNA is present with iron and H2O2, and so inhibiting DNA single strand breakage. These results suggest that the neuroprotective action of leucomentins is dependent on their ability to chelate iron.     

Protective effect of melatonin against in vitro iron ions and 7 mT 50 Hz magnetic field-induced DNA damage in rat lymphocytes

We have previously shown that simultaneous exposure of rat lymphocytes to iron ions and 50Hz magnetic field (MF) caused an increase in the number of cells with DNA strand breaks. Although the mechanism of MF-induced DNA damage is not known, we suppose that it involves free radicals. In the present study, to confirm our hypothesis, we have examined the effect of melatonin, an established free radicals scavenger, on DNA damage in rat peripheral blood lymphocytes exposed in vitro to iron ions and 50Hz MF. The alkaline comet assay was chosen for the assessment of DNA damage. During pre-incubation, part of the cell samples were supplemented with melatonin (0.5 or 1.0mM). The experiments were performed on the cell samples incubated for 3h in Helmholtz coils at 7mT 50Hz MF. During MF exposure, some samples were treated with ferrous chloride (FeCl2, 10microg/ml), while the rest served as controls. A significant increase in the number of cells with DNA damage was found only after simultaneous exposure of lymphocytes to FeCl2 and 7mT 50Hz MF, compared to the control samples or those incubated with FeCl2 alone. However, when the cells were treated with melatonin and then exposed to iron ions and 50Hz MF, the number of damaged cells was significantly reduced, and the effect depended on the concentration of melatonin. The reduction reached about 50% at 0.5mM and about 100% at 1.0mM. Our results indicate that melatonin provides protection against DNA damage in rat lymphocytes exposed in vitro to iron ions and 50Hz MF (7mT). Therefore, it can be suggested that free radicals may be involved in 50Hz magnetic field and iron ions-induced DNA damage in rat blood lymphocytes. The future experimental studies, in vitro and in vivo, should provide an answer to the question concerning the role of melatonin in the free radical processes in the power frequency magnetic field.     

NADPH- and NADH-dependent oxygen radical generation by rat liver nuclei in the presence of redox cycling agents and iron

Redox cycling agents such as paraquat and menadione increase the generation of reactive oxygen species in biological systems. The ability of NADPH and NADH to catalyze the generation of oxygen radicals from the metabolism of these redox cycling agents by rat liver nuclei was determined. The oxidation of hydroxyl radical scavenging agents by the nuclei was increased in the presence of menadione or paraquat, especially with NADPH as the reductant. Paraquat, even at high concentrations, was relatively ineffective with NADH. The highest rates of generation of .OH-like species occurred with ferric-EDTA as the iron catalyst. Certain ferric complexes such as ferric-ATP, ferric-citrate, or ferric ammonium sulfate, which were ineffective catalysts for .OH generation in the absence of paraquat or menadione, were reactive in the presence of the redox cycling agents. Oxidation of .OH scavengers was sensitive to catalase and competitive .OH-scavenging agents under all conditions. The redox cycling agents increased NADPH-dependent nuclear generation of H2O2; stimulation of H2O2 production may play a role in the increase in .OH generation by menadione and paraquat. Menadione inhibited nuclear lipid peroxidation, whereas paraquat and adriamycin were stimulatory. The nuclear lipid peroxidation with either NADPH or NADH plus the redox cycling agents was not sensitive to catalase or .OH scavengers. These results indicate that the interaction of rat liver nuclei with redox cycling agents and iron leads to the production of potent oxidants which initiate lipid peroxidation or oxidize .OH scavengers. Although NADPH is more effective, NADH can also participate in catalyzing the production of reactive oxygen intermediates from the interaction of quinone redox cycling agents with nuclei. The ability of redox cycling agents to interact with various ferric complexes to catalyze nuclear generation of potent oxidizing species with either NADPH or NADH as reductants may contribute to the oxidative stress, toxicity, and mutagenicity of these agents in biological systems.     

Biological activities of chemically synthesized analogues of the nonreducing sugar moiety of lipid A

Adriamycin-Fe3+ complex catalyzes the formation of hydroxyl radical from hydrogen peroxide but the DNA-adriamycin-iron ternary complex is much more effective. 11-Deoxyadriamycin, which shows no spectral evidence of complex formation with iron, was ineffective. The generation of hydroxyl radical by adriamycin-Fe3+ complex in the presence of DNA correlates with its ability to cleave DNA. Hydroxyl radicals are thus implicated as the reactive oxygen species involved in the DNA damage caused by the adriamycin-Fe3+ complex.     

Differential loss of enzyme activity by vitC and iron containing proteins

Our earlier studies showed that rabbit muscle phosphoglucomutase was irreversibly inactivated by exposure to a mixture of vitamin C, FeCl3 and O2. The enzyme lost about 70% of its phosphate (V.V. Desphande and J.G. Joshi, J. Biol. Chem. 260, 754-764, 1985). The present report shows that several other iron proteins can substitute for FeCl3 to a varying degree. The rate of inactivation by FeCl3 greater than ferritin greater than hemoglobin = hemerythrin greater than transferrin = ferridoxin = vitamin C. These iron compounds also produced dephosphoenzyme but did not dephosphorylate ATP, ADP, AMP or phospholipids.     

Endotoxin increases hepatic susceptibility to lipid peroxidation: a possible role of iron

The purpose of this study was to investigate the possible mechanism by which endotoxin enhances peroxidative damage to membrane lipids. Male B6C3 mice were treated with endotoxin intraperitoneally 0 or 20 mg/kg body weight for 24 h. Freshly prepared liver homogenate was incubated with either 1-5 mM of reduced glutathione (GSH), glucose, H(2)O(2), ascorbic acid (AA), FeSO(4), FeCl(3), EDTA, FeCl(3) plus AA, AA plus EDTA or EDTA plus FeCl(3) in phosphate-buffered saline (PBS), pH 7.0, or PBS, at 37 degrees C for 60 min. The levels of lipid peroxidation products, thiobarbituric acid reactants (TBAR), were significantly higher in the liver of endotoxin-treated mice, and the values were markedly increased following incubation. Compared to PBS, incubation with H(2)O(2), FeCl(3), FeSO(4), and AA, but not glucose, significantly enhanced TBAR formation. The greatest increase of TBAR was found when AA and FeCl(3) were added together. On the other hand, EDTA and GSH inhibited the formation of TBAR during incubation. When added before AA, EDTA completely inhibited the peroxidative effect of AA or FeSO4, and when added subsequent to AA, EDTA partially prevented the adverse effect of AA. The results obtained suggest that ionic iron plays an important role in initiating endotoxin-induced peroxidative damage to membrane lipids, and that AA may be involved in releasing iron from its protein complex and/or maintaining ionic iron in a reduced or catalytic state.     

Effect of Iron Chelators on Dopamine D2 Receptors

Nutritional iron deficiency induced in rats causes a selective reduction of [3H]spiperone binding in caudate nucleus. This effect can be reversed by iron supplementation in vivo. The possibility that iron may be involved in the dopamine D2 receptor was investigated by examining the effect of various iron and noniron chelators on the binding of [3H]spiperone in rat caudate nucleus. Iron chelators 1,10-phenanthroline, 2,4,6-tripyridyl-s-triazine, alpha, alpha'-dipyridyl, and desferrioxamine mesylate inhibited the binding of [3H]spiperone. The inhibition by 1,10-phenanthroline was noncompetitive and reversible. In the presence of FeCl2 or FeCl3, the inhibitory effect of 1,10-phenanthroline was potentiated. Iron salts or chelators were without effect on the binding of [3H]dihydroalprenolol to beta-adrenoreceptors in caudate nucleus; thus the action of iron chelators on the dopamine D2 receptor tends to be selective. Incubation of caudate nucleus membrane prepared from iron-deficient rats with FeCl2 or FeCl3 did not reverse the diminished binding of [3H]spiperone. The present study indicates that if iron is involved in the physiological regulation of dopamine D2 agonist-antagonist binding sites, it is more complex than hitherto considered.     

Interaction of ferric complexes with rat liver nuclei to catalyze NADH-and NADPH-Dependent production of oxygen radicals

The production of potent oxygen radicals by microsomal reaction systems has been well characterized. Relatively little attention has been paid to generation of oxygen radicals by liver nuclei, or to the interaction of nuclei with different ferric complexes to catalyze NADH- or NADPH-dependent production of reactive oxygen intermediates. Intact rat liver nuclei were capable of catalyzing an iron-dependent production of .OH as reflected by the oxidation of .OH scavenging agents such as 2-keto-4-thiomethylbutyrate, dimethyl sulfoxide, and t-butyl alcohol. Inhibition of .OH production by catalase implicates H2O2 as the precursor of .OH generated by the nuclei, whereas superoxide dismutase had only a partially inhibitory effect. The production of .OH with either cofactor was striking increased by addition of ferric-EDTA or ferric-diethylenetriamine-pentaacetic acid (DTPA) whereas ferric-ATP and ferric-citrate were not effective catalysts. All these ferric complexes were reduced by the nuclei in the presence of either NADPH or NADH. The pattern of iron chelate effectiveness in catalyzing lipid peroxidation by nuclei was opposite to that of .OH production; with either NADH or NADPH, nuclear lipid peroxidation was increased by the addition of ferric ammonium sulfate, ferric-ATP, or ferric-citrate, but not by ferric-EDTA or ferric-DTPA. NADPH-dependent nuclear lipid peroxidation was insensitive to catalase, superoxide dismutase, or .OH scavengers; the NADH-dependent reaction showed a partial sensitivity (30 to 40%) to these additions. The overall patterns of .OH production and lipid peroxidation by the nuclei are similar to those shown by microsomes, e.g., effect of ferric complexes, sensitivity to antioxidants; however, rates with the nuclei are less than 20% those of microsomes, which reflect the lower activities of NADPH- and NADH-cytochrome c reductase in the nuclei. The potential for nuclei to reduce ferric complexes and catalyze production of .OH-like species may play a role in the susceptibility of the genetic material to oxidative damage under certain conditions since such radicals would be produced site-directed and not exposed to cellular antioxidants.     

Kinetics of phosphate-mediated oxidation of ferrous iron and formation of 8-oxo-2'-deoxyguanosine in solutions of free 2'-deoxyguanosine and calf thymus DNA

Formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in solutions of free 2'-deoxyguanosine (dG) and calf thymus DNA (DNA) was compared for the diffusion-dependent and localised production of oxygen radicals from phosphate-mediated oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+). The oxidation of Fe2+ to Fe3+ was followed at 304 nm at pH 7.2 under aerobic conditions. Given that the concentration of Fe2+ >or=phosphate concentration, the rate of Fe2+ oxidation was significantly higher in DNA-phosphate as compared for the same concentration of inorganic phosphate. Phosphate catalysed oxidation of ferrous ions in solutions of dG or DNA led through the production of reactive oxygen species to the formation of 8-oxo-dG. The yield of 8-oxo-dG in solutions of dG or DNA correlated positively with the inorganic-/DNA-phosphate concentrations as well as with the concentrations of ferrous ions added. The yield of 8-oxo-dG per unit oxidised Fe2+ were similar for dG and DNA; thus, it differed markedly from radiation-induced 8-oxo-dG, where the yield in DNA was several fold higher.For DNA in solution, the localisation of the phosphate ferrous iron complex relative to the target is an important factor for the yield of 8-oxo-dG. This was supported from the observation that the yield of 8-oxo-dG in solutions of dG was significantly increased over that in DNA only when Fe2+ was oxidised in a high excess of inorganic phosphate (50 mM) and from the lower protection of DNA damage by the radical scavenger (hydroxymethyl)aminomethane (Tris)-HCl.     

UVA-induced oxidative damage to rat liver nuclei: reduction of iron ions and the relationship between lipid peroxidation and DNA damage

Lipid peroxidation and DNA damage and the relationship between the two events were studied in rat liver nuclei irradiated with low dose UVA. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) by spectrophotometric method and as malondialdehyde-TBA adduct by HPLC, and DNA damage was measured as 8-hydroxy-deoxyguanosine (8-OH-dGu) and strand breakage (or loss of double-stranded DNA) by a fluorometric analysis of alkaline DNA unwinding method. The results show that UVA irradiation by itself increased nuclear lipid peroxidation but caused little or no DNA strand breakage or 8-OH-dGu. When 0.5 mM ferric (Fe+3) or ferrous (Fe+2) ions were added to the nuclei during UVA irradiation, lipid peroxidation and DNA damage, measured both as 8-OH-dGu and loss of double-stranded DNA, were strongly enhanced. Lipid peroxidation occurred concurrently with the appearance of 8-OH-dGu. Fe3+ ions were reduced to Fe2+ in this UVA/Fe2+/nuclei system. Lipid peroxidation and DNA damage were neither inhibited by scavengers of hydroxyl radical and singlet oxygen nor inhibited by superoxide dismutase and catalase. Inclusion of EDTA or chain-breaking antioxidants, butylated hydroxytoluene (BHT) and diphenylamine (an alkoxy radical scavenger), inhibited lipid peroxidation but not the level of 8-OH-dGu. BHT also did not inhibit the loss of double-stranded DNA in this system. This study demonstrates the reduction of exogenous Fe+3 by UVA when added to rat liver nuclei, and, as a result, oxidative damage is strongly enhanced. In addition, the results show that DNA damage is not a result of lipid peroxidation in this UVA/Fe2+/nuclei system.     

Mitochondrial DNA Damage in Iron Overload

Chronic iron overload has slow and insidious effects on heart, liver, and other organs. Because iron-driven oxidation of most biologic materials (such as lipids and proteins) is readily repaired, this slow progression of organ damage implies some kind of biological “memory.” We hypothesized that cumulative iron-catalyzed oxidant damage to mtDNA might occur in iron overload, perhaps explaining the often lethal cardiac dysfunction. Real time PCR was used to examine the “intactness” of mttDNA in cultured H9c2 rat cardiac myocytes. After 3–5 days exposure to high iron, these cells exhibited damage to mtDNA reflected by diminished amounts of near full-length 15.9-kb PCR product with no change in the amounts of a 16.1-kb product from a nuclear gene. With the loss of intact mtDNA, cellular respiration declined and mRNAs for three electron transport chain subunits and 16 S rRNA encoded by mtDNA decreased, whereas no decrements were found in four subunits encoded by nuclear DNA. To examine the importance of the interactions of iron with metabolically generated reactive oxygen species, we compared the toxic effects of iron in wild-type and rho^o cells. In wild-type cells, elevated iron caused increased production of reactive oxygen species, cytostasis, and cell death, whereas the rho^o cells were unaffected. We conclude that long-term damage to cells and organs in iron-overload disorders involves interactions between iron and mitochondrial reactive oxygen species resulting in cumulative damage to mtDNA, impaired synthesis of respiratory chain subunits, and respiratory dysfunction.Patients with primary or secondary iron overload are liable to cardiac and hepatic failure, and type II diabetes. Iron is required for the activity of numerous iron- and heme-containing proteins, but “free” (i.e. redox active) iron catalyzes the formation of highly toxic reactive oxygen species (ROS)² that damage lipids, proteins, and DNA (1). This damage is assumed to arise from iron-catalyzed hydroxyl radical formation or, perhaps more likely, iron-centered radicals such as ferryl and perferryl (2, 3). Iron-driven oxidation events require that the metal interact with cellular oxidizing and reducing equivalents such as superoxide and hydrogen peroxide, a major source of which is “leak” of electrons from the mitochondrial electron transport chain (4–6).The present investigations were focused on the etiology of iron-mediated cardiac damage and specifically on the question of why, in patients with chronic iron overload, damage to organs such as the heart develops over a period of years, whereas most types of iron-mediated oxidation events can be repaired within minutes or hours. We have investigated the hypothesis that cumulative damage to DNA, specifically mtDNA, is critical to the slow development of cardiac dysfunction in chronic iron overload. In partial support of this idea, earlier studies clearly show that iron does promote DNA base oxidation as well as single and double strand DNA breaks. Mitochondrial DNA may be particularly vulnerable to such oxidation events inasmuch as it lacks histones, has less effective repair systems and, perhaps most importantly, resides within an organelle that ceaselessly generates ROS.Here, we report that, in cultured rat cardiac myocytes, iron overload causes (i) progressive loss of intact mtDNA, (ii) decreased expression of respiratory chain subunits encoded by mitochondrial, but not nuclear, DNA, and (iii) diminished respiratory function. Furthermore, it appears that iron-mediated cytotoxicity involves ROS generated by the mitochondrion itself because cells lacking mtDNA (and, therefore, respiration) are remarkably tolerant of iron overload. Overall, our results suggest that the slowly developing cardiac dysfunction seen in chronic iron overload arises secondary to cumulative iron-driven oxidant damage to mtDNA.     

Factors affecting catalase expression in Pseudomonas aeruginosa biofilms and planktonic cells

Previous work with Pseudomonas aeruginosa showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 microM as FeCl(3)) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.     

Iron binding of 3-hydroxychromone, 5-hydroxychromone, and sulfonated morin: Implications for the antioxidant activity of flavonols with competing metal binding sites

The iron binding properties and antioxidant activities of compounds with hydroxy-keto binding sites, 3-hydroxychromone, 5-hydroxychromone, and sulfonated morin were investigated. For these compounds, prevention of iron-mediated DNA damage and kinetics of Fe^II oxidation were studied in aqueous solutions close to physiological pH (pH 6). 3-Hydroxychromone and sulfonated morin inhibit iron-mediated DNA damage at lower concentrations than 5-hydroxychromone. All three compounds bind iron, but 3-hydroxychromone and sulfonated morin promote Fe^II oxidation much faster than 5-hydroxychromone. These results indicate that DNA damage inhibition by flavonols with competing hydroxy-keto binding sites is primarily due to iron binding at the 3-hydroxy-keto site. Iron oxidation rate also plays a significant role in antioxidant activity. In addition to iron binding and oxidation, reactive oxygen species scavenging occurs at high concentrations for the hydroxychromones. This study emphasizes the importance of iron binding in polyphenol antioxidant behavior and provides insights into the iron binding antioxidant activity of similar flavonols such as quercetin and myricetin.     

Hydrogen peroxide-induced apoptosis in HL-60 cells requires caspase-3 activation

Apoptosis has been associated with oxidative stress in biological systems. Caspases have been considered to play a pivotal role in the execution phase of apoptosis. However, which caspases function as executioners in reactive oxygen species (ROS)-induced apoptosis is not known. The present study was performed to identify the major caspases acting in ROS-induced apoptosis. Treatment of HL-60 cells with 50 μM hydrogen peroxide (H₂O₂) for 4 h induced the morphological changes such as condensed and/or fragmented nuclei, increase in caspase-3 subfamily protease activities, reduction of the procaspase-3 and a DNA fragmentation. To determine the role of caspases in H₂O₂-induced apoptosis, caspase inhibitors, acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone(Ac-YVAD-cmk), acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and acetyl-Val-Glu-lle-Aspaldehyde (Ac-VEID-CHO), selective for caspase-1 subfamily, caspase-3 subfamily and caspase-6, respectively, were loaded into the cells using an osmotic lysis of pinosomes method. Of these caspase inhibitors, only Ac-DEVD-CHO completely blocked morphological changes, caspase-3 subfamily protease activation and DNA ladder formation in H₂O₂-treated HL-60 cells. This inhibitory effect was dose-dependent. These results suggest that caspase-3, but not caspase-1 is required for commitment to ROS-triggered apoptosis.     

Prooxidant Action of Maltol: Role of Transition Metals in the Generation of Reactive Oxygen Species and Enhanced Formation of 8-hydroxy-2′-deoxyguanosine Formation in DNA

Maltol (3-hydroxy-2-methyl-4-pyrone) produced reactive oxygen species as a complex with transition metals. Maltol/iron complex inactivated aconitase the most sensitive enzyme to oxidative stress. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that the maltol/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Addition of maltol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2′-deoxyguanosine in DNA. Oxidation of ascorbic acid by CuSO₄ was effectively stimulated by addition of maltol, and the enhanced oxidation rate was markedly inhibited by the addition of catalase and superoxide dismutase. These results suggest that maltol can stimulate the copper reduction coupled with the oxidation of ascorbate, resulting in the production of superoxide radical which in turn converts to hydrogen peroxide and hydroxyl radical. Cytotoxic effect of maltol can be explained by its prooxidant properties: maltol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the production of hydroxyl radical causing the formation of DNA base adduct.     

Iron Oxide Nanoparticles Induce Oxidative Stress,DNA Damage,and Caspase Activation in the Human Breast Cancer Cell Line

Broad applications of iron oxide nanoparticles require an improved understanding of their potential effects on human health. In the present study, we explored the underlying mechanism through which iron oxide nanoparticles induce toxicity in human breast cancer cells (MCF-7). MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and lactate dehydrogenase assays were used to examine mechanisms of cytotoxicity. Concentration- and time-dependent cytotoxicity was observed in MCF-7 cells. Iron oxide nanoparticles were found to induce oxidative stress evidenced by the elevation of reactive oxygen species generation, lipid peroxidation, and depletion of superoxide dismutase, glutathione, and catalase activities in MCF-7 cells. Nuclear staining was performed using 4′, 6-diamidino-2-phenylindole (DAPI), and cells were analyzed with a fluorescence microscope. Iron oxide nanoparticles (60 μg/ml) induced substantial apoptosis that was identified by morphology, condensation, and fragmentation of the nuclei of the MCF-7 cells. It was also observed that the iron oxide NPs induced caspase-3 activity. DNA strand breakage was detected by comet assay, and it occurred in a concentration- and time-dependent manner. Thus, the data indicate that iron oxide nanoparticles induced cytotoxicity and genotoxicity in MCF-7 cells via oxidative stress. This study warrants more careful assessment of iron oxide nanoparticles before their industrial applications.