Ascorbic acid inhibits lysosomal autophagy of ferritin

Ascorbic acid retards ferritin degradation in K562 erythroleukemia cells leading to an increase in the availability of cellular iron (Bridges, K. R., and Hoffman, K. E. (1986) J. Biol. Chem. 261, 14273-14277). To explore the mechanism of this effect, the influence of ascorbate on subcellular ferritin distribution was examined. Cellular ferritin was pulse-labeled with 59Fe for 2 h, after which the cells were hypotonically lysed and fractionated on an 8% Percoll density gradient. Immediately after the labeling, all of the ferritin was in the cytoplasmic fractions at the top of the gradient. When the labeling was followed by a 24-h period of growth, a portion of the ferritin shifted to the lysosome-associated fractions at the bottom of the gradient, consistent with lysosomal autophagy of cytoplasmic ferritin. When ascorbate was added to the culture medium during the 24-h incubation, the magnitude of the shift was reduced. This process was also examined by size-fractionation of the contents of labeled cells using a Sepharose CL-6B column. Immediately after labeling, ferritin emerged from the column in two peaks, indicating the existence of both ferritin monomer and aggregates within the cytoplasm. After a 24-h period of growth, the monomer peak disappeared, while a new ferritin peak coincident with lysosomes emerged again, indicative of lysosomal autophagy of ferritin. In cells cultured with ascorbate for 24-h, there was a marked attenuation of the shift of ferritin to the lysosomal fractions. The monomer peak disappeared, as in the controls, but there was instead, an accumulation of ferritin as cytoplasmic aggregates. The total ferritin content of the ascorbate-treated cells was increased by 4-fold over that of the control. These experiments indicate that ascorbate blocks the degradation of cytoplasmic ferritin by reducing lysosomal autophagy of the protein. The access to the cell of the potentially toxic iron stored within the ferritin molecule is thereby increased.     

The active role of vitamin C in mammalian iron metabolism: Much more than just enhanced iron absorption!

Ascorbate is a cofactor in numerous metabolic reactions. Humans cannot synthesize ascorbate owing to inactivation of the gene encoding the enzyme l-gulono-γ-lactone oxidase, which is essential for ascorbate synthesis. Accumulating evidence strongly suggests that in addition to the known ability of dietary ascorbate to enhance nonheme iron absorption in the gut, ascorbate within mammalian systems can regulate cellular iron uptake and metabolism. Ascorbate modulates iron metabolism by stimulating ferritin synthesis, inhibiting lysosomal ferritin degradation, and decreasing cellular iron efflux. Furthermore, ascorbate cycling across the plasma membrane is responsible for ascorbate-stimulated iron uptake from low-molecular-weight iron–citrate complexes, which are prominent in the plasma of individuals with iron-overload disorders. Importantly, this iron-uptake pathway is of particular relevance to astrocyte brain iron metabolism and tissue iron loading in disorders such as hereditary hemochromatosis and β-thalassemia. Recent evidence also indicates that ascorbate is a novel modulator of the classical transferrin–iron uptake pathway, which provides almost all iron for cellular demands and erythropoiesis under physiological conditions. Ascorbate acts to stimulate transferrin-dependent iron uptake by an intracellular reductive mechanism, strongly suggesting that it may act to stimulate iron mobilization from the endosome. The ability of ascorbate to regulate transferrin iron uptake could help explain the metabolic defect that contributes to ascorbate-deficiency-induced anemia.     

Uptake of ferritin and iron bound to ferritin by rat hepatocytes: modulation by apotransferrin, iron chelators and chloroquine

Rat liver ferritin is an effective donor of iron to rat hepatocytes. Uptake of iron from ferritin by the cells is partially inhibited by including apotransferrin in the culture medium, but not by inclusion of diferric transferrin. This inhibition is dependent on the concentration of apotransferrin, with a 30% depression in iron incorporation in the cells detected at apotransferrin concentrations above 40 micrograms/ml. However, apotransferrin does not interfere with uptake of 125I-labeled ferritin, suggesting that apotransferrin decreases retention of iron taken up from ferritin by hepatocytes by sequestering a portion of released iron before it has entered the metabolic pathway of the cells. The iron chelators desferrioxamine (100 microM), citrate (10 mM) and diethylenetriaminepentaacetate (100 microM) reduce iron uptake by the cells by 35, 25 and 8%, respectively. In contrast, 1 mM ascorbate increases iron accumulation by 20%. At a subtoxic concentration of 100 microM, chloroquine depresses ferritin and iron uptake by hepatocytes by more than 50% after 3 h incubation. Chloroquine presumably acts by retarding lysosomal degradation of ferritin and recycling of ferritin receptors.     

Distinct mechanisms of ferritin delivery to lysosomes in iron-depleted and iron-replete cells

Ferritin is a cytosolic protein that stores excess iron, thereby protecting cells from iron toxicity. Ferritin-stored iron is believed to be utilized when cells become iron deficient; however, the mechanisms underlying the extraction of iron from ferritin have yet to be fully elucidated. Here, we demonstrate that ferritin is degraded in the lysosome under iron-depleted conditions and that the acidic environment of the lysosome is crucial for iron extraction from ferritin and utilization by cells. Ferritin was targeted for degradation in the lysosome even under iron-replete conditions in primary cells; however, the mechanisms underlying lysosomal targeting of ferritin were distinct under depleted and replete conditions. In iron-depleted cells, ferritin was targeted to the lysosome via a mechanism that involved autophagy. In contrast, lysosomal targeting of ferritin in iron-replete cells did not involve autophagy. The autophagy-independent pathway of ferritin delivery to lysosomes was deficient in several cancer-derived cells, and cancer-derived cell lines are more resistant to iron toxicity than primary cells. Collectively, these results suggest that ferritin trafficking may be differentially regulated by cell type and that loss of ferritin delivery to the lysosome under iron-replete conditions may be related to oncogenic cellular transformation.     

A simple assay for determination of iron release from ferritin in neuroblastoma cells.

A commercially available enzyme immunoassay was used to determine ferritin content and subsequently the loading and release of iron from ferritin in neuroblastoma cells. LS cells were incubated with 59Fe for 24 h, lysed, and the cytoplasmic ferritin was bound to monoclonal antibodies coupled to globules. After determination of the ferritin content the same globules with bound radioactive ferritin were measured in a gamma-counter. To illustrate the applicability of this test system, increased iron loading of cellular ferritin could be demonstrated in cycloheximide-treated cells; furthermore, release of iron was documented after incubation of LS cells with a combination of 6-hydroxydopamine and ascorbate. The assay turned out to be a simple method for determination of changes in 59Fe content of ferritin in neuroblastoma cells.     

Hepatic cellular distribution and degradation of iron oxide nanoparticles following single intravenous injection in rats: implications for magnetic resonance imaging

The purpose of this study was to determine the cellular distribution and degradation in rat liver following intravenous injection of superparamagnetic iron oxide nanoparticles used for magnetic resonance imaging (NC100150 Injection). Relaxometric and spectrophotometric methods were used to determine the concentration of the iron oxide nanoparticles and their degradation products in isolated rat liver parenchymal, endothelial and Kupffer cell fractions. An isolated cell phantom was also constructed to quantify the effect of the degradation products on the loss of MR signal in terms of decreased transverse relaxation times, T2*. The results of this study show that iron oxide nanoparticles found in the NC100150 Injection were taken up and distributed equally in both liver endothelial and Kupffer cells following a single 5 mg Fe/kg body wt. bolus injection in rats. Whereas endothelial and Kupffer cells exhibited similar rates of uptake and degradation, liver parenchymal cells did not take up the NC100150 Injection iron oxide particles. Light-microscopy methods did, however, indicate an increased iron load, presumably as ferritin/hemosiderin, within the hepatocytes 24 h post injection. The study also confirmed that compartmentalisation of ferritin/hemosiderin may cause a significant decrease in the MRI signal intensity of the liver. In conclusion, the combined results of this study imply that the prolonged presence of breakdown product in the liver may cause a prolonged imaging effect (in terms of signal loss) for a time period that significantly exceeds the half-life of NC100150 Injection iron oxide nanoparticles in liver.     

Ascorbate-mediated Iron Release from Ferritin in the Presence of Alloxan

Release of iron from ferritin requires reduction of ferric to ferrous iron. The iron can participate in the diabetogenic action of alloxan. We investigated the ability of ascorbate to catalyze the release of iron from ferritin in the presence of alloxan. Incubation of ferritin with ascorbate alone elicited iron release (33 nmol/10 min) and the generation of ascorbate free radical, suggesting a direct role for ascorbate in iron reduction. Iron release by ascorbate significantly increased in the presence of alloxan, but alloxan alone was unable to release measurable amounts of iron from ferritin. Superoxide dismutase significantly inhibited ascorbate-mediated iron release in the presence of alloxan, whereas catalase did not. The amount of alloxan radical (A·⁻) generated in reaction systems containing both ascorbate and alloxan decreased significantly upon addition of ferritin, suggesting that A·⁻ is directly involved in iron reduction. Although release of iron from ferritin and generation of A·⁻ were also observed in reactions containing GSH and alloxan, the amount of iron released in these reactions was not totally dependent on the amount of A·⁻ present, suggesting that other reductants in addition to A·⁻ (such as dialuric acid) may be involved in iron release mediated by GSH and alloxan. These results suggest that A·⁻ is the main reductant involved in ascorbate-mediated iron release from ferritin in the presence of alloxan and that both dialuric acid and A·⁻ contribute to GSH/alloxan-mediated iron release.     

Ferroportin-mediated mobilization of ferritin iron precedes ferritin degradation by the proteasome

Ferritin is a cytosolic molecule comprised of subunits that self-assemble into a nanocage capable of containing up to 4500 iron atoms. Iron stored within ferritin can be mobilized for use within cells or exported from cells. Expression of ferroportin (Fpn) results in export of cytosolic iron and ferritin degradation. Fpn-mediated iron loss from ferritin occurs in the cytosol and precedes ferritin degradation by the proteasome. Depletion of ferritin iron induces the monoubiquitination of ferritin subunits. Ubiquitination is not required for iron release but is required for disassembly of ferritin nanocages, which is followed by degradation of ferritin by the proteasome. Specific mammalian machinery is not required to extract iron from ferritin. Iron can be removed from ferritin when ferritin is expressed in Saccharomyces cerevisiae, which does not have endogenous ferritin. Expressed ferritin is monoubiquitinated and degraded by the proteasome. Exposure of ubiquitination defective mammalian cells to the iron chelator desferrioxamine leads to degradation of ferritin in the lysosome, which can be prevented by inhibitors of autophagy. Thus, ferritin degradation can occur through two different mechanisms.     

Mobilization of ferritin iron in erythroblasts by chelating agents

Intracellular ferritin in newt (Triturus cristatus) erythroblasts was accessible to the chelating effects of EDTA and pyridoxal phosphate. EDTA (0.5-1 mM) promoted release of radioactive iron from ferritin of pulse-labelled erythroblasts during chase incubation, but its continuous presence was not necessary for ferritin iron mobilization. Brief exposure to EDTA was sufficient to release 60-70% of ferritin 59Fe content during ensuing chase in EDTA-free medium. EDTA also suppressed cellular iron uptake and utilization for heme synthesis, but these activities were restored upon its removal. Pyridoxal-5'-phosphate (0.5-5 mM) also stimulated loss of radioactive iron from ferritin; however, ferritin iron release by pyridoxal phosphate required its continued presence. Unlike EDTA, pyridoxal phosphate did not interfere with iron uptake or its utilization for heme synthesis. Chelator-mobilized ferritin iron accumulated initially in the hemolysate as a low-molecular-weight component and appeared to be eventually released into the medium. No radioactive ferritin was found in the medium of chelator-treated cells, indicating that secretion or loss of ferritin was not responsible for decreasing cellular ferritin 59Fe content. Moreover, there was no transfer of radioactive iron between the low-molecular-weight component released into the medium and plasma transferrin. These results indicate that chelator-released ferritin iron is not available for cellular utilization in heme synthesis and that ferritin iron released by this process is not an alternative or complementary iron source for heme synthesis. Correlation of these data with effects of succinylacetone inhibition of heme synthesis and with previous studies indicates that the main role of erythroid cell ferritin is absorption and storage of excess iron not used for heme synthesis.     

Autophagy,ferritin and iron chelation

Ferritin is an iron storage molecule in vertebrates that stores iron in a redox inactive form. Ferritin is synthesized in response to high cellular iron levels and is degraded and iron released when iron demand is increased. Previously we determined that the turnover of ferritin occurs via the proteasome when the iron exporter ferroportin is expressed, and via the lysosome when the iron chelator deferoxamine is given to cells. Deferoxamine is used to treat hemochromatosis, a disease of iron accumulation that can be either genetic or acquired.

Autophagy provides a mechanism by which cytosolic proteins gain access to the lumen of lysosomes. Our results suggest that entry of ferritin into lysosomes is highly specific and not a consequence of generalized engulfment of cytosolic compartments by lysosomes. Entry of ferritin is also independent of the presence of LAMP-2, which suggests that ferritin entry does not result from chaperone-mediated autophagy. In summary, in this study we identify a new route that links ferritin degradation to activation of autophagy. The identification of this pathway will help to understand the molecular events that lead to activation of deferoxamine-mediated ferritin degradation and may contribute to the design of new therapeutic strategies for iron chelation therapy.     

The chemical form of mitochondrial iron in Friedreich's ataxia

Friedreich's ataxia (FRDA) results from cellular damage caused by a deficiency in the mitochondrial matrix protein frataxin. To address the effect of frataxin deficiency on mitochondrial iron chemistry, the heavy mitochondrial fraction (HMF) was isolated from primary fibroblasts from FRDA affected and unaffected individuals. X-ray absorption spectroscopy was used to characterize the chemical form of iron. Near K-edge spectra were fitted with a series of model iron compounds to determine the proportion of each iron species. Most of the iron in both affected and unaffected fibroblasts was ferrihydrite. The iron K-edge from unaffected HMFs were best fitted with poorly organized ferrihydrite modeled by frataxin whereas HMFs from affected cells were best fitted with highly organized ferrihydrite modeled by ferritin. Both had several minor iron species but these did not differ consistently with disease. Since the iron K-edge spectra of ferritin and frataxin are very similar, we present additional evidence for the presence of ferritin-bound iron in HMF. The predominant ferritin subunit in HMFs from affected cells resembled mitochondrial ferritin (MtFt) in size and antigenicity. Western blotting of native gels showed that HMF from affected cells had 3-fold more holoferritin containing stainable iron. We conclude that most of the iron in fibroblast HMF from both affected and unaffected cells is ferrihydrite but only FRDA affected cells mineralize significant iron in mitochondrial ferritin.     

Non-heme induction of heme oxygenase-1 does not alter cellular iron metabolism

The catabolism of heme is carried out by members of the heme oxygenase (HO) family. The products of heme catabolism by HO-1 are ferrous iron, biliverdin (subsequently converted to bilirubin), and carbon monoxide. In addition to its function in the recycling of hemoglobin iron, this microsomal enzyme has been shown to protect cells in various stress models. Implicit in the reports of HO-1 cytoprotection to date are its effects on the cellular handling of heme/iron. However, the limited amount of uncommitted heme in non-erythroid cells brings to question the source of substrate for this enzyme in non-hemolytic circumstances. In the present study, HO-1 was induced by either sodium arsenite (reactive oxygen species producer) or hemin or overexpressed in the murine macrophage-like cell line, RAW 264.7. Both of the inducers elicited an increase in active HO-1; however, only hemin exposure caused an increase in the synthesis rate of the iron storage protein, ferritin. This effect of hemin was the direct result of the liberation of iron from heme by HO. Cells stably overexpressing HO-1, although protected from oxidative stress, did not display elevated basal ferritin synthesis. However, these cells did exhibit an increase in ferritin synthesis, compared with untransfected controls, in response to hemin treatment, suggesting that heme levels, and not HO-1, limit cellular heme catabolism. Our results suggest that the protection of cells from oxidative insult afforded by HO-1 is not due to the catabolism of significant amounts of cellular heme as thought previously.     

Duodenal cytochrome b: a novel ferrireductase in airway epithelial cells

Catalytically active iron in the lung causes oxidative stress and promotes microbial growth that can be limited by intracellular sequestration of iron within ferritin. Because cellular iron uptake requires membrane ferrireductase activity that in the gut can be provided by duodenal cytochrome b (Dcytb), we sought Dcytb in the lung to test the hypothesis that it contributes to epithelial iron regulation by reducing Fe(3+) for cellular iron transport. Dcytb expression was found in respiratory epithelium in vitro and in vivo and was responsive to iron concentration. Iron transport was measured in human bronchial epithelial (HBE) cells using inductively coupled plasma atomic emission spectroscopy and was demonstrated to be partially inhibited in the presence of Dcytb-blocking antibody, suggesting that Dcytb reduces Fe(3+) for cellular iron transport. A definite source of reducing equivalents for Dcytb was sought but not identified. We found no evidence that ascorbate was involved but did demonstrate that O(2)(-). production decreased when Dcytb function was blocked. The presence of Dcytb in airway epithelial cells and its regulation by iron therefore may contribute to pulmonary cytoprotection.     

Superoxide ion as a primary reductant in ascorbate-mediated ferritin iron release

The mechanism of ascorbate-promoted ferritin iron reduction under aerobic conditions was studied. The initial rate of ferritin iron release was determined by spectrophotometric measurement of the Fe(ferrozine)3(2+) complex which absorbs at 562 nm. Variation of the initial ferrozine concentration had no influence on the rate of iron release suggesting that ferrozine does not participate in the rate-determining step. Experimental measurements of the initial rate of iron release as a function of ascorbate concentration resulted in saturation kinetics with Vmax = 2.0 X 10(-7) M.min-1 and KM = 1.3 X 10(-3) M. The effect of pH was quite pronounced with a maximal rate of iron release at pH 7.0. Stoichiometric measurements on the reaction mixture, with added catalase, resulted in a ratio of 2 Fe(II) released per ascorbate. Ascorbate-mediated iron release was inhibited 85% by superoxide dismutase, but 0% inhibition was noted with aposuperoxide dismutase. It is proposed that superoxide ion, generated during the iron-promoted oxidation of ascorbate, acts as a reductant of ferritin iron. A mechanism of ferritin iron release consistent with these experimental observations is discussed.     

Ultrastructure of ferritin in the leaves of <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> under stress conditions

The location and structure of ferritin in the parenchyma of leaf minor veins of the common ice plant (Mesembryanthemum crystallinum L.) treated with exogenous putrescine under salinity conditions were investigated by electron microscopy. Considerable aggregates of ferritin were detected in the chloroplasts of bundle sheath cells, in companion phloem cells, and other parenchyma cells of leaf minor veins. The structure of ferritin in the vascular parenchyma chloroplasts suggests that it was partially degraded and converted to phytosiderin. This point of view is based on indistinct structure of Fe-containing cores of ferritin molecules, break of distance between the cores, and their pronounced ability to aggregate and produce larger structures. Aggregation of Fe-containing cores apparently pointed to the destruction of ferritin protein envelope or its partial degradation. In a certain stage of ferritin destruction, electron-dense material and the structures resembling small vesicles appeared between the Fe-containing cores. Electron-dense inclusions, whose structure was similar to that of phytosiderin, were also detected in the vacuoles. Examination of the cross sections done without additional staining showed that the same as ferritin, phytosiderin in the chloroplasts and vacuoles was dark-colored against weakly colored cellular structures. In the vascular parenchyma of control plant leaves, the level of ferritin and phytosiderin was greater than in the mesophyll and much lower than in the plants simultaneously treated with NaCl and putrescine. In control material, iron cores of ferritin and phytosiderin were more light-colored and 2–3 times smaller in size than in the experimental treatment. Destruction of ferritin essentially did not occur in the mesophyll but was observed in the chloroplasts of bundle sheath cells on the border between the mesophyll and vascular bundle. The presence of much ferritin and phytosiderin on the border between the mesophyll and the vessels is accounted for by the fact that the vascular parenchyma is a buffer area that maintains a specific concentration of iron in the mesophyll of leaves and other parts of the plant. Within the cell, the role of such a buffer is performed by ferritin and vacuoles. Transformation of ferritin to insoluble hydrophobic phytosiderin is supposed to be an efficient way of withdrawing the excess of active iron from the cellular metabolism and therefore of relaxing oxidative stress. Ferritin and phytosiderin were detected not only in parenchyma cells of leaf minor veins but in sieve tubes as well. This suggests that iron may be transported within the plant as a component of protein complex.     

Protective effect of serotonin on 6-hydroxydopamine- and dopamine-induced oxidative damage of brain mitochondria and synaptosomes and PC12 cells

The present study elucidated the effects of indoleamines (serotonin, melatonin, and tryptophan) on oxidative damage of brain mitochondria and synaptosomes induced either by 6-hydroxydopamine (6-OHDA) or by iron plus ascorbate and on viability loss in dopamine-treated PC12 cells. Serotonin (1-100 microM), melatonin (100 microM), and antioxidant enzymes attenuated the effects of 6-OHDA, iron plus ascorbate, or 1-methyl-4-phenylpyridinium on mitochondrial swelling and membrane potential formation. Serotonin and melatonin decreased the attenuation of synaptosomal Ca(2+) uptake induced by either 6-OHDA alone or iron plus ascorbate. Serotonin and melatonin inhibited the production of reactive oxygen species, formation of malondialdehyde and carbonyls, and thiol oxidation in mitochondria and synaptosomes and decreased degradation of 2-deoxy-D-ribose. Unlike serotonin, melatonin did not reduce the iron plus ascorbate-induced thiol oxidation. Tryptophan decreased thiol oxidation and 2-deoxy-D-ribose degradation but did not inhibit the production of reactive oxygen species and formation of oxidation products in the brain tissues. Serotonin and melatonin attenuated the dopamine-induced viability loss, including apoptosis, in PC12 cells. The results suggest that serotonin may attenuate the oxidative damage of mitochondria and synaptosomes and the dopamine-induced viability loss in PC12 cells by a decomposing action on reactive oxygen species and inhibition of thiol oxidation and shows the effect comparable to melatonin. Serotonin may show a prominent protective effect on the iron-mediated neuronal damage.     

Antioxidant capacity of parsley cells (Petroselinum crispum L.) in relation to iron-induced ferritin levels and static magnetic field

This study was aimed to evaluate antioxidant response of parsley cells to 21 ppm iron and static magnetic field (SMF; 30 mT). The activity of catalase (CAT) and ascorbate peroxidase (APX) and the contents of malonyldialdehyde, iron and ferritin were measured at 6 and 12 h after treatments. Exposure to SMF increased the activity of CAT in treated cells, while combination of iron and SMF treatments as well as iron supply alone decreased CAT activity, compared to that of control cells. Combination of SMF with iron treatment reduced iron content of the cells and ameliorated mal effect of iron on CAT activity. All treatments reduced APX activity; however, the content of total ascorbate increased in response to iron and SMF+iron. The results showed that among the components of antioxidant system of parsley cells, enhanced activity of CAT in SMF-treated cells and increase of ascorbate in SMF+Fe-treated ones were responsible for the maintenance of membranes integrity. Ferritin contents of SMF- and SMF+Fe-treated cells also decreased significantly 12 h after treatments, compared to those of the control cells. These results cast doubt on the proposed functions of ferritin as a putative reactive oxygen species detoxifying molecule.     

Iron supplementation and oxidative damage to DNA in healthy individuals with high plasma ascorbate

Previously, we have investigated the potential for a pro-oxidant interaction of iron and ascorbate in vivo in iron and ascorbate cosupplementation or ascorbate supplementation studies. In this study, for the first time, the effects of iron supplementation on oxidative damage to DNA in healthy individuals with plasma ascorbate levels at the upper end of the normal range were examined. Forty female and male volunteers (mean plasma ascorbate approximately equal to 70 micromol/L) were supplemented with a daily dose of syrup (ferrous glycine sulphate equivalent to 12.5 mg iron) for 6 weeks. Serum ferritin, transferrin bound iron, % saturation of transferrin and plasma ascorbate were assessed and the mean dietary intakes of all subjects were estimated through food frequency questionnaires. Oxidative damage to DNA bases from white blood cells was measured by gas chromatography/mass spectrometry with selected-ion monitoring (GC/MS-SIM), using isotope-labelled standards for quantification. Iron supplementation did not affect any of the iron status parameters. There were also no detrimental effects, over the period under investigation, in terms of oxidative damage to DNA. However, the effects of larger doses or of longer supplementation periods should also be investigated.     

Deleterious iron-mediated oxidation of biomolecules

Iron is an essential metal for most biological organisms. However, if not tightly controlled, iron can mediate the deleterious oxidation of biomolecules. This review focuses on the current understanding of the role of iron in the deleterious oxidation of various biomolecules, including DNA, protein, lipid, and small molecules, e.g., ascorbate and biogenic amines. The effect of chelation on the reactivity of iron is also addressed, in addition to iron-associated toxicities. The roles of the iron storage protein ferritin as both a source of iron for iron-mediated oxidations and as a mechanism to safely store iron in cells is also addressed.