Oxidative stress mediates toxicity of pyridoxal isonicotinoyl hydrazone analogs

Pyridoxal isonicotinoyl hydrazone (PIH) and many of its analogs are effective iron chelators in vivo and in vitro, and are of interest for the treatment of secondary iron overload. Because previous work has implicated the Fe(3+)-chelator complexes as a determinant of toxicity, the role of iron-based oxidative stress in the toxicity of PIH analogs was assessed. The Fe(3+) complexes of PIH analogs were reduced by K562 cells and the physiological reductant, ascorbate. Depletion of the antioxidant, glutathione, sensitized Jurkat T lymphocytes to the toxicity of PIH analogs and their Fe(3+) complexes, and toxicity of the chelators increased with oxygen tension. Fe(3+) complexes of pyridoxal benzoyl hydrazone (PBH) and salicyloyl isonicotinoyl hydrazone (SIH) caused lipid peroxidation and toxicity in K562 cells loaded with eicosapentenoic acid (EPA), a readily oxidized fatty acid, whereas Fe(PIH)(2) did not. The lipophilic antioxidant, vitamin E, completely prevented both the toxicity and lipid peroxidation caused by Fe(PBH)(2) in EPA-loaded cells, indicating a causal relationship between oxidative stress and toxicity. PBH also caused concomitant lipid peroxidation and toxicity in EPA-loaded cells, both of which were reversed as its concentration increased. In contrast, PIH was inactive, while SIH was equally toxic toward control and EPA-loaded cells, without causing lipid peroxidation, indicating a much smaller contribution of oxidative stress to the mechanism of toxicity of these analogs. In summary, PIH analogs and their Fe(3+) complexes are redox active in the intracellular environment. The contribution of oxidative stress to the overall mechanism of toxicity varies across the series.     

Intracellular iron trafficking: role of cytosolic ligands

Iron acquired by cells is delivered to mitochondria for metabolic processing via pathways comprising undefined chemical forms. In order to assess cytosolic factors that affect those iron delivery pathways, we relied on microscopy and flow-cytometry for monitoring iron traffic in: (a) K562 erythroleukemia cells labeled with fluorescent metal-sensors targeted to either cytosol or mitochondria and responsive to changes in labile iron and (b) permeabilized cells that retained metabolically active mitochondria accessible to test substrates. Iron supplied to intact cells as transferrin-Fe(III) or Fe(II)-salts evoked concurrent metal ingress to cytosol and mitochondria. With either supplementation modality, iron ingress into cytosol was mostly absorbed by preloaded chelators, but ingress into mitochondria was fully inhibited only by some chelators, indicating different cytosol-to-mitochondria delivery mechanisms. Iron ingress into cytosol or mitochondria were essentially unaffected by depletion of cytosolic iron ligands like glutathione or the hypothesized 2,5 dihydroxybenzoate (2,5-DHBA) siderophore/chaperone. These ligands also failed to affect mitochondrial iron ingress in permeabilized K562 cells suspended in cytosol-simulating medium. In such medium, mitochondrial iron uptake was >6-eightfold higher for Fe(II) versus Fe(III), showed saturable properties and submicromolar K(1/2) corresponding to cytosolic labile iron levels. When measured in iron(II)-containing media, ligands like AMP, ADP or ATP, did not affect mitochondrial iron uptake whereas in iron(III)-containing media ADP and ATP reduced it and AMP stimulated it. Thus, cytosolic iron forms demonstrably contribute to mitochondrial iron delivery, are apparently not associated with DHBA analogs or glutathione but rather with resident components of the cytosolic labile iron pool.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on *OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

The effect of various chelating agents on the mobilization of iron from reticulocytes in the presence and absence of pyridoxal isonicotinoyl hydrazone

The chelating agent pyridoxal isonicotinoyl hydrazone (PIH) has recently been shown to mobilize ⁵⁹Fe from reticulocytes loaded with non-heme ⁵⁹Fe. In this study, various chelating agents were tested for their ability to effect the mobilization of iron from reticulocytes by PIH. They fall into several groups. The largest group includes chelators such as citrate, ethylenediaminetetracetic acid and desferrioxamine, which fail to affect PIH-induced iron mobilization and do not mobilize iron per se. Either these chelators do not enter reticulocytes or they do not take up iron from PIH-Fe complexes. The second group includes chelators such as 2,2′-bipyridine, 1,10-phenanthroline, bathophenanthroline sulfonate and N,N′-ethylenebis(o-hydroxyphenylglycine) which inhibit PIH-induced iron mobilization from reticulocytes and, when added together with PIH, induce radioiron accumulation in an alcohol-soluble fraction of reticulocytes. It appears that these chelators enter the cell and compete with PIH for ⁵⁹Fe(II), but having bound iron are unable to cross the cell membrane. Spectral analysis suggests that Fe(II) chelators such as 2,2′-bipyridine and 1,10-phenanthroline remove iron from Fe(II)PIH but are not able to do so from Fe(III)PIH. Then there are compounds such as 2,3-dihydroxybenzoic acid and catechol which potentiate PIH-induced iron mobilization although they are unable to mobilize iron from reticulocytes by themselves. Lastly, there is a group of miscellaneous compounds which include chelators that either potentiate the iron-mobilizing effect of PIH as well as mobilizing iron from reticulocytes by themselves (tropolone), or that reduce PIH-induced iron mobilization while themselves having an iron-mobilizing effect (N,N′-bis(2,3-dihydroxybenzoyl)-1,6-diaminohexane). In further experiments, heme was found to stimulate globin synthesis in reticulocytes, the heme synthesis of which was inhibited by PIH, suggesting that PIH is probably not toxic to the cells.     

Cytotoxic iron chelators: characterization of the structure,solution chemistry and redox activity of ligands and iron complexes of the di-2-pyridyl ketone isonicotinoyl hydrazone (<Emphasis Type="Bold">H</Emphasis>PKIH) analogues

Di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) and a range of its analogues comprise a series of monobasic acids that are capable of binding iron (Fe) as tridentate (N,N,O) ligands. Recently, we have shown that these chelators are highly cytotoxic, but show selective activity against cancer cells. Particularly interesting was the fact that cytotoxicity of the HPKIH analogues is maintained even after complexation with Fe. To understand the potent anti-tumor activity of these compounds, we have fully characterized their chemical properties. This included examination of the solution chemistry and X-ray crystal structures of both the ligands and Fe complexes from this class and the ability of these complexes to mediate redox reactions. Potentiometric titrations demonstrated that all chelators are present predominantly in their charge-neutral form at physiological pH (7.4), allowing access across biological membranes. Keto–enol tautomerism of the ligands was identified, with the tautomers exhibiting distinctly different protonation constants. Interestingly, the chelators form low-spin (diamagnetic) divalent Fe complexes in solution. The chelators form distorted octahedral complexes with Fe^II, with two tridentate ligands arranged in a meridional fashion. Electrochemistry of the Fe complexes in both aqueous and non-aqueous solutions revealed that the complexes are oxidized to their ferric form at relatively high potentials, but this oxidation is coupled to a rapid reaction with water to form a hydrated (carbinolamine) derivative, leading to irreversible electrochemistry. The Fe complexes of the HPKIH analogues caused marked DNA degradation in the presence of hydrogen peroxide. This observation confirms that Fe complexes from the HPKIH series mediate Fenton chemistry and do not repel DNA. Collectively, studies on the solution chemistry and structure of these HPKIH analogues indicate that they can bind cellular Fe and enhance its redox activity, resulting in oxidative damage to vital biomolecules.Electronic Supplementary Material Supplementary material is available in the online version of this article at .Abbreviations DFO desferrioxamine - HPKIH di-2-pyridyl ketone isonicotinoyl hydrazone - HNIH 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone - HPCIH 2-pyridinecarbaldehyde isonicotinoyl hydrazone - HPIH pyridoxal isonicotinoyl hydrazone - L linear DNA - OC open circular DNA - SC supercoiled DNA     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on ⋅OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as β-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against ^⋅OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for ^⋅OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of ^⋅OH from H₂O₂. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H₂O₂. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on ^⋅OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

Uptake and intracellular distribution of iron from transferrin and chelators in erythroid cells

Summary Iron chelators of different physicochemical properties were studied for their ability to donate iron in vitro to uninduced K562 cells, human bone marrow cells and purified human erythroblasts. To a large extent uptake was found to be related to lipophilicity and those chelators able to deliver iron to the cells in significant amounts were also able to deliver iron to ferritin and haem. Some differences in the distribution of iron delivered was observed but no chelator showed exclusive delivery to or rejection of a particular cellular iron compartment. Several chelators could probably substitute for transferrin and be used to probe metabolic events subsequent to iron removal from transferrin. Two chelators which were excellent iron donors were also found to cause considerable inhibition of iron incorporation into haem from transferrin. The implications of this for in vivo toxicity are briefly discussed.     

Acidosis Potentiates Oxidative Neuronal Death by Multiple Mechanisms

Both acidosis and oxidative stress contribute to ischemic brain injury. The present study examines interactions between acidosis and oxidative stress in murine cortical cultures. Acidosis (pH 6.2) was found to potentiate markedly neuronal death induced by H2O2 exposure. To determine if this effect was mediated by decreased antioxidant capacity at low pH, the activities of several antioxidant enzymes were measured. Acidosis was found to reduce the activities of glutathione peroxidase and glutathione S-transferase by 50-60% (p < 0.001) and the activity of glutathione reductase by 20% (p < 0.01) in lysates of the cortical cultures. Like acidosis, direct inhibition of glutathione peroxidase with mercaptosuccinate also potentiated H2O2 toxicity. Because acidosis may accelerate hydroxyl radical production by the Fenton reaction, the effect of iron chelators was also examined. Both desferrioxamine and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, two structurally different iron chelators, significantly reduced H2O2-induced neuronal death under both pH 7.2 and pH 6.2 conditions. These results suggest that the increased cell death produced by severe acidosis during cerebral ischemia may result in part from exacerbation of oxidative injury. This exacerbation may result from both impaired antioxidant enzyme functions and increased intracellular free iron levels.     

Green tea polyphenol epigallocatechin-3-gallate protects HepG2 cells against CYP2E1-dependent toxicity

Chronic ethanol consumption causes oxidative damage in the liver, and induction of cytochrome P450 2E1 (CYP2E1) is one pathway involved in oxidative stress produced by ethanol. The hepatic accumulation of iron and polyunsaturated fatty acids significantly contributes to ethanol hepatotoxicity in the intragastric infusion model of ethanol treatment. The objective of this study was to analyze the effect of the green tea flavanol epigallocatechin-3-gallate (EGCG), which has been shown to prevent alcohol-induced liver damage, on CYP2E1-mediated toxicity in HepG2 cells overexpressing CYP2E1 (E47 cells). Treatment of E47 cells with arachidonic acid plus iron (AA + Fe) was previously reported to produce synergistic toxicity in E47 cells by a mechanism dependent on CYP2E1 activity and involving oxidative stress and lipid peroxidation. EGCG protected E47 cells against toxicity and loss of viability induced by AA+Fe; EGCG had no effect on CYP2E1 activity. Prevention of this toxicity was associated with a reduction in oxidative damage as reflected by decreased generation of reactive oxygen species, a decrease in lipid peroxidation, and maintenance of intracellular glutathione in cells challenged by AA+Fe in the presence of EGCG. AA+Fe treatment caused a decline in the mitochondrial membrane potential, which was also blocked by EGCG. In conclusion, EGCG exerts a protective action on CYP2E1-dependent oxidative stress and toxicity that may contribute to preventing alcohol-induced liver injury, and may be useful in preventing toxicity by various hepatotoxins activated by CYP2E1 to reactive intermediates.     

Characteristics of iron release from isolated heavy and light endosomes

1. Endosomes were isolated from K 562 cells after 3 min after the endocytosis of a single cohort of transferrin molecules. 2. The change in 125I/59Fe ratio of heavy and light endosomes, relative to that of the transferrin used and 59Fe from light endosomes. 3. Incubation of heavy and light endosomes with PBS or PIH showed equal ATP specific iron release from both heavy and light endosomes, but in the presence of a NADH/NAD+ redox couple iron release from light endosomes was reduced. 4. Incubation of heavy and light endosomes with PIH and NEM did not completely abolish ATP specific iron release from heavy and light endosomes.     

Comparison of various iron chelators and prochelators as protective agents against cardiomyocyte oxidative injury

Oxidative stress is a common denominator of numerous cardiovascular disorders. Free cellular iron catalyzes the formation of highly toxic hydroxyl radicals, and iron chelation may thus be an effective therapeutic approach. However, using classical iron chelators in diseases without iron overload poses risks that necessitate more advanced approaches, such as prochelators that are activated to chelate iron only under disease-specific oxidative stress conditions. In this study, three cell-membrane-permeable iron chelators (clinically used deferasirox and experimental SIH and HAPI) and five boronate-masked prochelator analogs were evaluated for their ability to protect cardiac cells against oxidative injury induced by hydrogen peroxide. Whereas the deferasirox-derived agents TIP and TRA-IMM displayed negligible protection and even considerable toxicity, the aroylhydrazone prochelators BHAPI and BSIH-PD provided significant cytoprotection and displayed lower toxicity after prolonged cellular exposure compared to their parent chelators HAPI and SIH, respectively. Overall, the most favorable properties in terms of protective efficiency and low inherent cytotoxicity were observed with the aroylhydrazone prochelator BSIH. BSIH efficiently protected both H9c2 rat cardiomyoblast-derived cells and isolated primary rat cardiomyocytes against hydrogen peroxide-induced mitochondrial and lysosomal dysregulation and cell death. At the same time, BSIH was nontoxic at concentrations up to its solubility limit (600 μM) and in 72-h incubation. Hence, BSIH merits further investigation for prevention and/or treatment of cardiovascular disorders associated with a known (or presumed) component of oxidative stress.     

Effect of iron chelators on the transferrin receptor in K562 cells

Delivery of iron to K562 cells by diferric transferrin involves a cycle of binding to surface receptors, internalization into an acidic compartment, transfer of iron to ferritin, and release of apotransferrin from the cell. To evaluate potential feedback effects of iron on this system, we exposed cells to iron chelators and monitored the activity of the transferrin receptor. In the present study, we found that chelation of extracellular iron by the hydrophilic chelators desferrioxamine B, diethylenetriaminepentaacetic acid, or apolactoferrin enhanced the release from the cells of previously internalized 125I-transferrin. Presaturation of these compounds with iron blocked this effect. These chelators did not affect the uptake of iron from transferrin. In contrast, the hydrophobic chelator 2,2-bipyridine, which partitions into cell membranes, completely blocked iron uptake by chelating the iron during its transfer across the membrane. The 2,2-bipyridine did not, however, enhance the release of 125I-transferrin from the cells, indicating that extracellular iron chelation is the key to this effect. Desferrioxamine, unlike the other hydrophilic chelators, can enter the cell and chelate an intracellular pool of iron. This produced a parallel increase in surface and intracellular transferrin receptors, reaching 2-fold at 24 h and 3-fold at 48 h. This increase in receptor number required ongoing protein synthesis and could be blocked by cycloheximide. Diethylenetriaminepentaacetic acid or desferrioxamine presaturated with iron did not induce new transferrin receptors. The new receptors were functionally active and produced an increase in 59Fe uptake from 59Fe-transferrin. We conclude that the transferrin receptor in the K562 cell is regulated in part by chelatable iron: chelation of extracellular iron enhances the release of apotransferrin from the cell, while chelation of an intracellular iron pool results in the biosynthesis of new receptors.     

Partition coefficients of the iron (III) complexes of pyridoxal isonicotinoyl hydrazone and its analogs and the correlation to iron chelation efficacy. Correction of some reported partition coefficients

Pyridoxal isonicotinoyl hydrazone (PIH), salicylaldehydebenzoyl hydrazone (SBH), and their analogschelate iron(III) and show promise asorally effective drugs for treating diseases of iron overload. Theirbiological activity isrelated to their lipophilicity, as measured by their partition coefficients P betweenn-octanoland water. However, the method of calculating log P described in an article in this journal(Edwardet al. 1995; BioMetals, 8, 209-217) is faulty for compounds such as PIH, SBH andtheir analogs whichcontain adjacent hydrophilic groups. Consequently, the calculations reportedin the article, based on erro-neouslog P values of the chelating molecules, giveerroneous log P values of the iron(III) complexes. Thechelators most effective inmobilizing 59 Fe from reticulocytes have log P < 2.8, not log P < 0 and theiron(III)complexes of the most effective chelators have log P < 3.1, not log P < 0.     

Hydrolysis of pyridoxal isonicotinoyl hydrazone and its analogs

An orally available iron chelator is desirable for the treatment of secondary iron overload. Pyridoxal isonicotinoyl hydrazone (PIH) and its analogs effectively mobilize iron in vivo and in vitro, and are therefore promising candidates for this purpose. PIH analogs undergo significant amino acid-catalyzed hydrolysis in cell culture medium and in serum, achieving equilibrium with their corresponding aldehydes and hydrazides with half-times of 1-8 h. The extent of hydrolysis in RPMI is significant, even in experiments of a few hours' duration, although the half-life of PIH in phosphate-buffered saline (PBS) is approximately 24 h. Therefore, the biological effects (e.g., 59Fe mobilization, toxicity) of these iron chelators have been underestimated in previous studies. Measurement of the affinity of PIH analogs for Fe(3+) under conditions in which hydrolysis is minimal resulted in conditional affinity constants of 10(26) to 10(27) M, which are much lower than predicted by the overall formation constants determined under conditions that likely allowed extensive hydrolysis. These data indicate the importance of hydrolysis of PIH analogs in the interpretation of previous studies, and the importance of designing clinically useful analogs whose efficacies are not limited by hydrolysis.     

Metabolic depletion inhibits the uptake of nontransferrin-bound iron by K562 cells

The effect of metabolic inhibitors on nontransferrin bound iron transport by K562 cells was investigated. Incubation with 1 microM rotenone, 10 microM antimycin, or 0.5 mM 2,4-dinitrophenol effectively reduced ATP levels by approximately 50%. Both the rate and extent of Fe+3 uptake were impaired in ATP-depleted cells, which display a reduced Vmax for uptake. K562 cell ferrireductase activity was also lowered by metabolic inhibitors, suggesting that the apparent energy requirements for transport reside in the reduction of Fe+3 to Fe+2. However, ATP depletion was found to inhibit the rate and extent of Fe+2 uptake as well. Thus, the transbilayer passage of Fe+2 and/or Fe+3 appears to be an energy-requiring process. These features possibly reflect properties of the transport mechanism associated with a recently identified K562 cell transport protein, called SFT for "Stimulator of Fe Transport," since exogenous expression of its activity is also affected by ATP depletion.     

Opposing roles for ERK1/2 in neuronal oxidative toxicity: distinct mechanisms of ERK1/2 action at early versus late phases of oxidative stress

Glutamate-induced oxidative toxicity is mediated by glutathione depletion in the HT22 mouse hippocampal cell line. Previous results with pharmacological agents implicated the extracellular signal-regulated kinases-1/2 (ERK1/2) in glutamate toxicity in HT22 cells and immature embryonic rat cortical neurons. In this report, we definitively establish a role for ERK1/2 in oxidative toxicity using dominant negative MEK1 expression in transiently transfected HT22 cells to block glutamate-induced cell death. In contrast, chronic activation of ERK (i.e. brought about by transfection of constitutively active ERK2 chimera) is not sufficient to trigger HT22 cell death demonstrating that ERK1/2 activation is not sufficient for toxicity. Activation of ERK1/2 in HT22 cells has a distinct kinetic profile with an initial peak occurring between 30 min and 1 h of glutamate treatment and a second peak typically emerging after 6 h. We demonstrate here that the initial phase of ERK1/2 induction is because of activation of metabotropic glutamate receptor type I (mGluRI). ERK1/2 activation by mGluRI contributes to an HT22 cell adaptive response to oxidative stress as glutamate-induced toxicity is enhanced upon pharmacological inhibition of mGluRI. The protective effect of ERK1/2 activation at early times after glutamate treatment is mediated by a restoration of glutathione (GSH) levels that are reduced because of depletion of intracellular cysteine pools. Thus, ERK1/2 appears to play dual roles in HT22 cells acting as part of a cellular adaptive response during the initial phases of glutamate-induced oxidative stress and contributing to toxicity during later stages of stress.     

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.     

Role of an oxidative stress in the macrophage dysfunction caused by erythrophagocytosis

A phagocytic challenge with immunoglobulin G (IgG)-coated erythrocytes (EIgGs) has been shown to cause a subsequent depression of macrophage respiratory burst capacity and phagocytic function. The present study evaluated the hypothesis that this macrophage dysfunction is caused by an oxidative stress. An oxidative stress induced by ferric ammonium citrate (FAC) plus cumene hydroperoxide (CHP) caused a depression of macrophage function that was attenuated by antioxidants and iron chelators. In contrast, the same antioxidants and iron chelators did not alter changes caused by a challenge with EIgGs. EIgG challenge caused an increase in lipid peroxidation but failed to deplete glutathione (GSH) or decrease the activity of glyceraldehyde-3-phosphate dehydrogenase (GA-3-PD), suggesting that there was only a slight oxidative stress. Inhibition of the Fc gamma receptor (Fc gammaR) stimulated respiratory burst by removing calcium during the challenge did not attenuate the changes caused by an EIgG challenge. A phagocytic challenge with nonerythrocyte particles, IgG-coated beads (BIgGs), did not depress the respiratory burst capacity but did depress phagocytic function. Fc gammaR expression was depressed following a phagocytic challenge but not an oxidative stress. Thus, an oxidative stress can depress macrophage function, but the dysfunction caused by a phagocytic challenge with EIgGs involves Fc gammaR depletion and the erythrocyte contents rather than an oxidative stress.     

Effects of Oxidative Stress Caused by Hyperoxia and Diquat. A Study in Isolated Hepatocytes

The effects of oxidative stress caused by hyperoxia or administration of the redox active compound diquat were studied in isolated hepatocytes, and the relative contribution of lipid peroxidation, glutathione (GSH) depletion, and NADPH oxidation to the cytotoxicity of active oxygen species was investigated.

The redox cycling of diquat occurred primarily in the microsomal fraction since diquat was found not ' to penetrate into the mitochondria. Depletion of intracellular GSH by pretreatment of the animals with diethyl maleate promoted lipid peroxidation and sensitized the cells to oxidative stress. Diquat toxicity was also greatly enhanced when glutathione reductase was inhibited by pretreatment of the cells with 1,3-bis(2-chloroethyI)-1-nitrosourea. Despite extensive lipid peroxidation, loss of cell viability was not observed, with either hyperoxia or diquat, until the GSH level had fallen below ≈ 6 nmol/10⁶ cells.

The iron chelator desferrioxamine provided complete protection against both diquat-induced lipid peroxidation and loss of cell viability. In contrast, the antioxidant a-tocopherol inhibited lipid peroxidation but provided only partial protection from toxicity. The hydroxy! radical scavenger α-keto-γ-methiol butyric acid, finally, also provided partial protection against diquat toxicity but had no effect on lipid peroxidation.

The results indicate that there is a critical GSH level above which cell death due to oxidative stress is not observed. As long as the glutathione peroxidase – glutathione reductase system is unaffected, even relatively low amounts of GSH can protect the cells by supporting glutathione peroxidase-mediated metabolism of H₂O₂ and lipid hydroperoxides.