Iron chelators reduce chromosomal breaks in ataxia-telangiectasia cells

Ataxia-telangiectasia (A-T) is characterized by ataxia, genomic instability, and increased cancer incidence. Previously, iron chelator concentrations which suppressed normal cell colony formation increased A-T cell colony formation. Similarly, iron chelators preferentially increased A-T cell colony formation following peroxide exposure compared to normal cells. Last, A-T cells exhibited increased short-term sensitivity to labile iron exposure compared to normal cells, an event corrected by recombinant ATM (rATM) expression. Since chromosomal damage is important in A-T pathology and iron chelators exert beneficial effects on A-T cells, we hypothesized that iron chelators would reduce A-T cell chromosomal breaks. We treated A-T, normal, and A-T cells expressing rATM with labile iron, iron chelators, antioxidants, and t-butyl hydroperoxide, and examined chromosomal breaks and ATM activation. Additionally, the effect of ATM-deficiency on transferrin receptor (TfR) expression and TfR activity blockage in A-T and syngeneic A-T cells expressing rATM was examined. We report that (1) iron chelators and iron-free media reduce spontaneous and t-butyl hydroperoxide-induced chromosomal breaks in A-T, but not normal, or A-T cells expressing rATM; (2) labile iron exposure induces A-T cell chromosomal breaks, an event lessened with rATM expression; (3) desferal, labile iron, and copper activate ATM; (4) A-T cell TfR expression is lowered with rATM expression and (5) blocking TfR activity with anti-TfR antibodies increases A-T cell colony formation, while lowering chromosomal breaks. ATM therefore functions in iron responses and the maintenance of genomic stability following labile iron exposure.     

Desferrioxamine treatment increases the genomic stability of Ataxia-telangiectasia cells

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by genomic instability, chronic oxidative damage, and increased cancer incidence. Compared to normal cells, AT cells exhibit unusual sensitivity to exogenous oxidants, including t-butyl hydroperoxide (t-BOOH). Since ferritin releases labile iron under oxidative stress (which is chronic in AT) and labile iron mediates the toxic effects of t-butyl hydroperoxide, we hypothesized that chelation of intracellular labile iron would increase the genomic stability of AT cells, with and without exogenous oxidative stress. Here we report that desferrioxamine treatment increases the plating efficiency of AT, but not normal cells, in the colony forming-efficiency assay (a method often used to measure genomic stability). Additionally, desferrioxamine increases AT, but not normal cell resistance, to t-butyl hydroperoxide in this assay. Last, AT cells exhibit increased sensitivity to the toxic effects of FeCl(2) in the colony forming-efficiency assay and fail to demonstrate a FeCl(2)-induced G(2) checkpoint response when compared to normal cells. Our data indicates that: (1) chelation of labile iron increases genomic stability in AT cells, but not normal cells; and (2) AT cells exhibit deficits in their responses to iron toxicity. While preliminary, our findings suggest that AT might be, in part, a disorder of iron metabolism and treatment of individuals with AT with desferrioxamine might have clinical efficacy.     

Iron loading and oxidative stress in the Atm-/- mouse liver

Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder resulting in a myriad of abnormalities, including progressive neurodegeneration and cancer predisposition. At the cellular level, A-T is a disease of chronic oxidative stress (OS) causing damage to proteins, lipids, and DNA. OS is contributed to by pro-oxidative transition metals such as iron that catalyze the conversion of weakly reactive oxygen species to highly reactive hydroxyl radicals. Iron-associated OS has been linked to neurodegeneration in Alzheimer's and Parkinson's diseases and development of lymphoid tumors (which afflict ～30% of A-T patients). To investigate iron regulation in A-T, iron indexes, regulatory genes, and OS markers were studied in livers of wild-type and Ataxia telangiectasia mutated (Atm) null mice on control or high-iron diets. Atm(-/-) mice had increased serum iron, hepatic iron, and ferritin and significantly higher Hepcidin compared with wild-type mice. When challenged with the high-iron diet, Bmp6 and Hfe expression was significantly increased. Atm(-/-) mice had increased protein tyrosine nitration and significantly higher Heme Oxygenase (decycling) 1 levels that were substantially increased by a high-iron diet. Ferroportin gene expression was significantly increased; however, protein levels were unchanged. We demonstrate that Atm(-/-) mice have a propensity to accumulate iron that is associated with a significant increase in hepatic OS. The iron-induced increase in hepcidin peptide in turn suppresses ferroportin protein levels, thus nullifying the upregulation of mRNA expression in response to increased OS. Our results suggest that increased iron status may contribute to the chronic OS seen in A-T patients and development of disease pathology.     

Iron oxide particles for molecular magnetic resonance imaging cause transient oxidative stress in rat macrophages

Iron oxide particles are a promising marker in molecular magnetic resonance imaging. They are used to label distinct cell populations either in vitro or in vivo. We investigated for the first time whether small citrate-coated very small superparamagnetic iron oxide particles (VSOPs) can lead to an increase in cellular oxidative stress. We incubated rat macrophages (RAW) in vitro with iron oxide particles. We observed a massive uptake of VSOPs measured both with atomic absorption spectroscopy and with NMR, which could be visualized by confocal laser scanning microscopy. After incubation, cells were lysed and the levels of malonyldialdehyde (MDA) and protein carbonyls were determined. We found a significant increase in both MDA and protein carbonyl levels after incubation with the particles. Surprisingly, 24 h after incubation, a significant indication of oxidative stress could no longer be observed. The increase in oxidative stress seems to be transient and closely linked to the incubation procedure. The iron chelator desferal and the intracellular spin trap PBN caused a significant reduction in oxidative stress to almost control levels. This indicates that the augmentation of oxidative stress is closely linked to the free iron during incubation. Proliferation assays showed that incorporation of VSOPs did not lead to long-term cytotoxic effects even though the iron oxide particles remained in the cell. Magnetic labeling of cells with VSOPs seems to cause transient oxidative conditions not affecting cellular viability and seems to be a usable approach for molecular magnetic resonance imaging.     

Foam cell death induced by 7beta-hydroxycholesterol is mediated by labile iron-driven oxidative injury: mechanisms underlying induction of ferritin in human atheroma

Human atherosclerotic lesions typically contain large amounts of ferritin associated with apoptotic macrophages and foam cells, although the reasons are unknown. In the present investigation, we studied the relationship between ferritin induction and occurrence of apoptosis in 7beta-hydroxycholesterol (7beta-OH)-treated monocytic cells and macrophages. We found that 7beta-OH enlarges the intracellular labile iron pool, increases formation of reactive oxygen species (ROS), and induces ferritin and cytosolic accumulation of lipid droplets, lysosomal destabilization, and apoptototic macrophage death. Since ferritin is a phase II-type protective protein, our findings suggest that ferritin upregulation here worked as an inefficient defense mechanism. Addition to the culture medium of both a membrane-permeable iron chelator 10-phenanthroline and the non-membrane-permeable iron chelators apoferritin and desferrioxamine afforded significant protection against the 7beta-OH-induced effects. Consequently, endocytosed iron compounds dramatically augmented 7beta-OH-induced cytotoxicity. We conclude that oxidized lipid 7beta-OH causes not only foam cell formation but also oxidative damage with abnormal metabolism of cellular iron. The findings suggest that modulation of iron metabolism in human atheroma may be a potential therapeutic strategy against atherosclerosis.     

Iron-induced oxidative stress up-regulates calreticulin levels in intestinal epithelial (Caco-2) cells

Calreticulin, a molecular chaperone involved in the folding of endoplasmic reticulum synthesized proteins, is also a shock protein induced by heat, food deprivation, and chemical stress. Mobilferrin, a cytosolic isoform of calreticulin, has been proposed to be an iron carrier for iron recently incoming into intestinal cells. To test the hypothesis that iron could affect calreticulin expression, we investigated the possible associations of calreticulin with iron metabolism. To that end, using Caco-2 cells as a model of intestinal epithelium, the mass and mRNA levels of calreticulin were evaluated as a function of the iron concentration in the culture media. Increasing the iron content in the culture from 1 to 20 microM produced an increase in calreticulin mRNA and a two-fold increase in calreticulin. Increasing iron also induced oxidative damage to proteins, as assessed by the formation of 4-hydroxy-2-nonenal adducts. Co-culture of cells with the antioxidants quercetin, dimethyltiourea and N-acetyl cysteine abolished both the iron-induced oxidative damage and the iron-induced increase in calreticulin. We postulate that the iron-induced expression of calreticulin is part of the cellular response to oxidative stress generated by iron.     

Quercetin as a shuttle for labile iron

The antioxidant activity of flavonoids may involve their ability to complex body iron in non-redox-active forms. In this study, it was found that the catechol flavonoids rutin and quercetin are able to suppress redox-active labile plasma iron (LPI) in both buffered solution and in iron-overloaded sera. Both flavonoids are effective in loading the metal into the iron-transport protein transferrin. Iron derivatives of quercetin and rutin are able to permeate cell membranes, however, only free quercetin is able to gain access to the cytosol and decrease intracellular labile iron pools. These results suggest that the antioxidant activity of quercetin may be dependent on its ability to shuttle labile iron from cell compartments followed by its transfer to transferrin.     

Aminoacetone induces loss of ferritin ferroxidase and iron uptake activities

Aminoacetone (AA) is a threonine and glycine metabolite overproduced and recently implicated as a contributing source of methylglyoxal (MG) in conditions of ketosis. Oxidation of AA to MG, NH4+, and H

2

O

2

has been reported to be catalyzed by a copper-dependent semicarbazide sensitive amine oxidase (SSAO) as well as by copper- and iron ion-catalyzed reactions with oxygen. We previously demonstrated that AA-generated O2•al (AA

•

) induce dose-dependent Fe(II) release from horse spleen ferritin (HoSF); no reaction occurs under nitrogen. In the present study we further explored the mechanism of iron release and the effect of AA on the ferritin apoprotein. Iron chelators such as EDTA, ATP and citrate, and phosphate accelerated AA-promoted iron release from HoSF, which was faster in horse spleen isoferritins containing larger amounts of phosphate in the core. Incubation of apoferritin with AA (2.5-50 mM, after 6 h) changes the apoprotein electrophoretic behavior, suggesting a structural modification of the apoprotein by AA-generated ROS. Superoxide dismutase (SOD) was able to partially protect apoferritin from structural modification whereas catalase, ethanol, and mannitol were ineffective in protection. Incubation of apoferritin with AA (1-10 mM) produced a dose-dependent decrease in tryptophan fluorescence (13-30%, after 5 h), and a partial depletion of protein thiols (29% after 24 h). The AA promoted damage to apoferritin produced a 40% decrease in apoprotein ferroxidase activity and an 80% decrease in its iron uptake ability. The current findings of changes in ferritin and apoferritin may contribute to intracellular iron-induced oxidative stress during AA formation in ketosis and diabetes mellitus.     

Transient increase of the labile iron pool in HepG2 cells by intravenous iron preparations.

Intravenous iron, used for the treatment of anemia in chronic renal failure and other diseases, represents a possible source of free iron in tissue cells, particularly in the liver. In this study we examined the effect of different sources of intravenous iron (IVI) on the labile iron pool (LIP) which represents the nonferritin-bound, redox-active iron that is implicated in oxidative stress and cell injury. Furthermore, we examined the role of the LIP for the synthesis of ferritin. We used HepG2 cells as a well known model for hepatoma cells and monitored the LIP with the metal-sensitive fluorescent probe, calcein-AM, the fluorescence of which is quenched on binding to iron. We showed that steady state LIP levels in HepG2 cells were increased transiently, up to three-fold compared to control cells, as an adaptive response to long-term IVI exposure. In relation to the amount of iron in the LIP, the ferritin levels increased and the iron content of ferritin decreased. As any fluctuation in the LIP, even when it is only transient (e.g. after exposure to intravenous iron in this study), may result either in impairment of synthesis of iron containing proteins or in cell injury by pro-oxidants. Such findings in nonreticuloendothelial cells may have important implications in the generation of the adverse effects of chronic iron exposure reported in dialysis patients.     

Mitochondrial models of pathologies with oxidative stress. Efficiency of alkalization to reduce mitochondrial damage

Previously, we developed a method to monitor the development of oxidative stress in isolated liver mitochondria. The method is based on recording of membrane potential changes in response to sequential introduction of low concentrations (5–20 μM) of tert-butyl hydroperoxide (tBHP). It allows monitoring of the extent of amplification or attenuation of oxidative stress caused by external influences (changes in incubation conditions, additions of biologically active substances). Based on this method, we created a mitochondrial model for the study and improvement of treatment of pathologies associated with oxidative stress. The following two processes were simulated in the experiments: 1) introduction of desferal for treatment of serious diseases caused by cell overload with iron (high desferal concentrations were shown to suppress mitochondrial energetics); 2) efficiency of alkalization to reduce mitochondrial damage induced by oxidative stress. The experiments have shown that even a small increase in pH (alkalization) increases the amount of tBHP that can be added to mitochondria before the MPTP (“mitochondrial permeability transition pore”) is induced. The effect of alkalization was shown to be close to the effect of cyclosporin A in the pH range 7.2–7.8. The mechanism of the similarities of these effects in the organism and in mitochondrial suspensions is explained by the increase in toxic reactive oxygen species in both systems under oxidative stress.     

On the Cytoprotective Role of Ferritin in Macrophages and its Ability to Enhance Lysosomal Stability

Macrophages have a great capacity to take up (eg. by endocytosis and phagocytosis) exogenous sources of iron which could potentially become cytotoxic, particularly following the intralysosomal formation of low-molecular weight, redox active iron, and under conditions of oxidative stress. Following autophago-cytosis of endogenous ferritin/apoferritin, these compounds may serve as chelators of such lysosomal iron and counteract the occurrence of iron-mediated intralysosomal oxidative reactions. Such redox-reactions have been shown to lead to destabilisation of lysosomal membranes and result in leakage of damaging lysosomal contents to the cytosol. In this study we have shown: (i) human monocyte-derived macrophages to accumulate ferritin in response to iron exposure; (ii) iron to destabilise macrophage secondary lysosomes when the cells are exposed to H₂O₂; and (iii) endocytosed apoferritin to act as a stabiliser of the acidic vacuolar compartment of iron-loaded macrophages. While the endogenous ferritin accumulation which was induced by iron exposure was not sufficient to protect cells from the damaging effects of H₂O₂, exogenously added apoferritin, as well as the potent iron chelator desferrioxamine, afforded significant protection. It is suggested that intralysosomal formation of haemosiderin, from partially degraded ferritin, is a protective strategy to suppress intralysosomal iron-catalysed redox reactions. However, under conditions of severe macrophage lysosomal iron-overload, induction of ferritin synthesis is not enough to completely prevent the enhanced cytotoxic effects of H₂O₂.     

Dysregulation of hepatic iron with aging: implications for heat stress-induced oxidative liver injury

Environmental heat stress is associated with an age-related increase in hepatic oxidative damage and an exaggerated state of oxidative stress. The purpose of this investigation was to evaluate the regulation of hepatic iron after heat stress. A secondary aim was to determine a potential role for iron in heat stress-induced liver injury. Hyperthermia-induced alterations in hepatic iron were evaluated in young (6 mo) and old (24 mo) Fischer 344 rats by exposing them to a two-heat stress protocol. Livers were harvested at several time points after the second heating and assayed for labile and nonheme iron. In the control condition, there was no difference in labile iron between age groups. Both labile iron and storage iron were not altered by hyperthermia in young rats, but both were increased immediately after heating in old rats. To evaluate a role for iron in liver injury, hepatic iron content was manipulated in young and old rats, and then both groups were exposed to heat stress. Iron administration to young rats significantly increased hepatic iron content and ferritin but did not affect markers of lipid peroxidation under control conditions or after heat stress. In old rats, iron chelation with deferoxamine prevented the increase in nonheme iron, labile iron, ferritin, and lipid peroxidation after heat stress. These results suggest that iron may play a role in hepatic injury after hyperthermia. Thus, dysregulation of iron may contribute to the gradual decline in cellular and physiological function that occurs with aging.     

Differential response of iron metabolism to oxidative stress generated by antimycin A and nitrofurantoin

The close interrelationship of oxidative stress and iron is evident by the influence of intracellular reactive oxygen species on iron metabolism. Oxygen radicals can lead to release of iron from iron-sulfur proteins and ferritin, and can damage iron-containing enzymes such as mitochondrial aconitase. Treatment of HepG2 human hepatoma cells with antimycin A has two effects relating to iron depending on the concentrations of antimycin A: increase of the labile iron pool and stimulation of non-transferrin-bound iron uptake. Whereas the first could also be generated with nitrofurantoin, the stimulation of non-transferrin-bound iron uptake was only seen with antimycin A and needed considerably higher concentrations. Pretreatment of the cells with ebselen, which scavenges peroxides, reverted only the effect of nitrofurantoin on the labile iron pool. Depletion with iron chelators before or after treatment with antimycin A diminished the stimulation of non-transferrin-bound iron uptake. We conclude that the generation of oxygen radicals in the mitochondria leads to the liberation of iron from mitochondrial enzymes, which enters the labile iron pool. But high concentrations of antimycin A leading to the stimulation of non-transferrin-bound iron uptake is possibly not related to the inhibition of the respiratory chain.     

Protein nitration increased by simulated weightlessness and decreased by melatonin and quercetin in PC12 cells.

A variety of experiments suggest that space flight is associated with an increase in oxidative stress in organism. To explore the effects of oxidative stress on neuronal cells during microgravity, we used rat pheochromocytoma (PC12) cells as a neuronal cell model, cultured in a clinostat, which could simulate microgravity, to investigate the effects of reactive nitrogen species on protein nitration in PC12 cells during clinorotation. The effects of melatonin and quercetin on protein nitration in PC12 cells were also assayed to evaluate the possible protective role of melatonin or quercetin as an antioxidant. The results of immunological staining showed that after the 3 days' clinorotation the protein expressions of neuronal nitric oxide synthase and inducible nitric oxide synthesis were up-regulated. Our data also reflected that the concentrations of nitric oxide and nitrotyrosine were significantly increased after clinorotation, and they were reduced markedly in cells that were treated with 50 micromol/L melatonin or 0.5 micromol/L quercetin during simulated microgravity, when compared to those of control cells. These results suggest that clinorotation-induced weightlessness increases oxidative stress responses in PC12 cells, and melatonin or quercetin was shown to protect PC12 cells from oxidative damage during simulated weightlessness.     

Desferal inhibits breast tumor growth and does not interfere with the tumoricidal activity of doxorubicin

Desferal is a clinically approved iron chelator used to treat iron overload. Doxorubicin is an anthracycline cancer chemotherapy drug used in the treatment of breast cancer. It can undergo redox cycling in the presence of iron to produce reactive oxygen species. The oxidant-generating activity of doxorubicin is thought to be responsible for the cardiotoxic side effects of the drug, but it is unclear whether it is also required for its anti-tumor activity. To test whether an iron-chelating antioxidant would interfere with the tumor-killing activity of doxorubicin, nude mice were transplanted with xenografts of human breast cancer MDA-MB 231 cells and then treated with doxorubicin and/or desferal. Not only did desferal not interfere with the anti-tumor activity of doxorubicin, it inhibited tumor growth on its own. In vitro studies confirmed that desferal inhibits breast tumor growth. However, it did not induce apoptosis, nor did it induce cell cycle arrest. Instead, desferal caused cytostasis, apparently through iron depletion. The cytostatic activity of desferal was partially ameliorated by pretreatment with iron-saturated transferrin, and transferrin receptor expression on breast cancer cells nearly doubled after exposure to desferal. In contrast to its effect on tumor cells, desferal did not inhibit growth of normal breast epithelial cells. The data indicate that the anti-tumor activity of doxorubicin is not dependent on iron-mediated ROS production. Furthermore, desferal may have utility as an adjunctive chemotherapy due to its ability to inhibit breast tumor growth and cardiotoxic side effects without compromising the tumor-killing activity of an anthracycline chemotherapy drug.     

Quercetin Protects against Okadaic Acid-Induced Injury via MAPK and PI3K/Akt/GSK3β Signaling Pathways in HT22 Hippocampal Neurons

Increasing evidence shows that oxidative stress and the hyperphosphorylation of tau protein play essential roles in the progression of Alzheimer’s disease (AD). Quercetin is a major flavonoid that has anti-oxidant, anti-cancer and anti-inflammatory properties. We investigated the neuroprotective effects of quercetin to HT22 cells (a cell line from mouse hippocampal neurons). We found that Okadaic acid (OA) induced the hyperphosphorylation of tau protein at Ser199, Ser396, Thr205, and Thr231 and produced oxidative stress to the HT22 cells. The oxidative stress suppressed the cell viability and decreased the levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), mitochondria membrane potential (MMP) and Glutathione peroxidase (GSH-Px). It up-regulated malondialdehyde (MDA) production and intracellular reactive oxygen species (ROS). In addition, phosphoinositide 3 kinase/protein kinase B/Glycogen synthase kinase3β (PI3K/Akt/GSK3β) and mitogen activated protein kinase (MAPK) were also involved in this process. We found that pre-treatment with quercetin can inhibited OA-induced the hyperphosphorylation of tau protein and oxidative stress. Moreover, pre-treatment with quercetin not only inhibited OA-induced apoptosis via the reduction of Bax, and up-regulation of cleaved caspase 3, but also via the inhibition of PI3K/Akt/GSK3β, MAPKs and activation of NF-κB p65. Our findings suggest the therapeutic potential of quercetin to treat AD.     

Modulation of Paraoxonase 2 (PON2) in Mouse Brain by the Polyphenol Quercetin: A Mechanism of Neuroprotection?

Quercetin is a common flavonoid polyphenol which has been shown to exert neuroprotective actions in vitro and in vivo. Though quercetin has antioxidant properties, it has been suggested that neuroprotection may be ascribed to its ability of inducing the cell’s own defense mechanisms. The present study investigated whether quercetin could increase the levels of paraoxonase 2 (PON2), a mitochondrial enzyme expressed in brain cells, which has been shown to have potent antioxidant properties. PON2 protein, mRNA, and lactonase activity were highest in mouse striatal astrocytes. Quercetin increased PON2 levels, possibly by activating the JNK/AP-1 pathway. The increased PON2 levels induced by quercetin resulted in decreased oxidative stress and ensuing toxicity induced by two oxidants. The neuroprotective effect of quercetin was significantly diminished in cells from PON2 knockout mice. These findings suggest that induction of PON2 by quercetin represents an important mechanism by which this polyphenol may exert its neuroprotective action.     

Redox regulation and oxidant activation of heme oxygenase-1

The ultraviolet A (UVA, 320–400 nm) component of sunlight has the potential to generate an oxidative stress in cells and tissue so that antioxidants (both endogenous and exogenous) strongly influence the biological effects of UVA. The expression of several genes (including heme oxygenase-1, HO-1; collagenase; the CL100 phosphatase and the nuclear oncogenes, c-fos and c-jun) is induced following physiological doses of UVA to cells and this effect can be strongly enhanced by removing intracellular glutathione or enhancing singlet oxygen lifetime. We have observed that heme is released from microsomal heme-containing proteins by UVA and other oxidants and that activation of HO-1 expression by UVA correlates with levels of heme release. UVA radiation also leads to an increase in labile iron pools (either directly or via HO-1) and eventual increases in ferritin levels. The role of heme oxygenase in protection of skin fibroblasts is probably an emergency inducible defense pathway to remove heme liberated by oxidants. The slower increase in ferritin levels is an adaptive response which serves to keep labile iron pools low and thereby reduce Fenton chemistry and oxidant-induced chain reactions involving lipid peroxidation. In keratinocytes, the primary target of UVA radiation, heme oxygenase levels are constitutively high (because of HO-2 expression). Since there is a corresponding increase in basal levels of ferritin the epidermis appears to be well protected constitutively against the oxidative stress generated by UVA.