Assay of Fe(III) Chelate Reductase Activity in Arabidopsis thaliana Root.

A sensitive FerroZine assay is used to measure the membrane-bound ferric-chelate reductase activity in the Arabidopsis thaliana roots. In Arabidopsis, FRO2 (FERRIC CHELATE REDUCTASE 2) encodes the Fe(III) chelate reductase and its expression is induced by iron deficiency. As FRO2 reduces Fe(III) to soluble Fe(II), the resulting Fe(II) forms a purple-colored complex with the dye FerroZine. The concentration of the Fe(II)-FerroZine is directly proportional to the absorbance at 562 nm.

Cell Labeling with Magneto-Endosymbionts and the Dissection of the Subcellular Location, Fate, and Host Cell Interactions.

PURPOSE: The purposes of this study are to characterize magneto-endosymbiont (ME) labeling of mammalian cells and to discern the subcellular fate of these living contrast agents. MEs are novel magnetic resonance imaging (MRI) contrast agents that are being used for cell tracking studies. Understanding the fate of MEs in host cells is valuable for designing in vivo cell tracking experiments. PROCEDURES: The ME's surface epitopes, contrast-producing paramagnetic magnetosomal iron, and genome were studied using immunocytochemistry (ICC), Fe and MRI contrast measurements, and quantitative polymerase chain reaction (qPCR), respectively. These assays, coupled with other common assays, enabled validation of ME cell labeling and dissection of ME subcellular processing. RESULTS: The assays mentioned above provide qualitative and quantitative assessments of cell labeling, the subcellular localization and the fate of MEs. ICC results, with an ME-specific antibody, qualitatively shows homogenous labeling with MEs. The ferrozine assay shows that MEs have an average of 7 fg Fe/ME, ∼30 % of which contributes to MRI contrast and ME-labeled MDA-MB-231 (MDA-231) cells generally have 2.4 pg Fe/cell, implying ∼350 MEs/cell. Adjusting the concentration of Fe in the ME growth media reduces the concentration of non-MRI contrast-producing Fe. Results from the qPCR assay, which quantifies ME genomes in labeled cells, shows that processing of MEs begins within 24 h in MDA-231 cells. ICC results suggest this intracellular digestion of MEs occurs by the lysosomal degradation pathway. MEs coated with listeriolysin O (LLO) are able to escape the primary phagosome, but subsequently co-localize with LC3, an autophagy-associated molecule, and are processed for digestion. In embryos, where autophagy is transiently suppressed, MEs show an increased capacity for survival and even replication. Finally, transmission electron microscopy (TEM) of ME-labeled MDA-231 cells confirms that the magnetosomes (the MRI contrast-producing particles) remain intact and enable in vivo cell tracking. CONCLUSIONS: MEs are used to label mammalian cells for the purpose of cell tracking in vivo, with MRI. Various assays described herein (ICC, ferrozine, and qPCR) allow qualitative and quantitative assessments of labeling efficiency and provide a detailed understanding of subcellular processing of MEs. In some cell types, MEs are digested, but the MRI-producing particles remain. Coating with LLO allows MEs to escape the primary phagosome, enhances retention slightly, and confirms that MEs are ultimately processed by autophagy. Numerous intracellular bacteria and all endosymbiotically derived organelles have evolved molecular mechanisms to avoid intracellular clearance, and identification of the specific processes involved in ME clearance provides a framework on which to develop MEs with enhanced retention in mammalian cells.

Is Iron Chelation Important in Preventing Glycation of Bovine Serum Albumin in Vitro?

The role of metal (especially) iron ions has been postulated to play a prominent role in protein glycation, suggesting antiglycating effectiveness of metal chelators. However, this rule may not apply to all model glycation systems. We found that metal chelators are not effective in prevention of glycation of bovine serum albumin (BSA) in vitro, and there is no correlation between the antiglycating effects of 32 compounds and their iron chelation activity as measured with the ferrozine test. These data indicate that the glycation of BSA in vitro is iron-independent and is not a proper system to study the role of metals in protein glycation.

A novel antimycobacterial compound acts as an intracellular iron chelator.

Efficient iron acquisition is crucial for the pathogenesis of Mycobacterium tuberculosis. Mycobacterial iron uptake and metabolism are therefore attractive targets for antitubercular drug development. Resistance mutations against a novel pyrazolopyrimidinone compound (PZP) that is active against M. tuberculosis have been identified within the gene cluster encoding the ESX-3 type VII secretion system. ESX-3 is required for mycobacterial iron acquisition through the mycobactin siderophore pathway, which could indicate that PZP restricts mycobacterial growth by targeting ESX-3 and thus iron uptake. Surprisingly, we show that ESX-3 is not the cellular target of the compound. We demonstrate that PZP indeed targets iron metabolism; however, we found that instead of inhibiting uptake of iron, PZP acts as an iron chelator, and we present evidence that the compound restricts mycobacterial growth by chelating intrabacterial iron. Thus, we have unraveled the unexpected mechanism of a novel antimycobacterial compound.

Interference of ferric ions with ferrous iron quantification using the ferrozine assay.

The ferrozine assay is a widely used colorimetric method for determining soluble iron concentrations. We provide evidence for a heretofore unrecognized interference of ferric ions (Fe(3+)) on ferrous iron (Fe(2+)) measurements performed in the dark. Fe(3+) concentrations affected the absorbance measurements, which linearly increased with incubation time.

Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway.

Myeloperoxidase (MPO) is a heme-containing enzyme that generates hypochlorous acid (HOCl) from chloride (Cl(-)) and hydrogen peroxide (H2O2). It is implicated in the pathology of several chronic inflammatory conditions such as cardiovascular and pulmonary diseases and cancer. Recently we have shown that HOCl can destroy the heme prosthetic group of hemoproteins. Here, we investigated whether the HOCl formed during steady-state catalysis is able to destroy the MPO heme moiety and thereby function as a major source of free iron. UV-visible spectra and H2O2-specific electrode measurements recorded during steady-state HOCl synthesis by MPO showed that the degree of MPO heme destruction increased after multiple additions of H2O2 (10 µM), precluding the enzyme from functioning at maximum activity (80-90% inhibition). MPO heme destruction occurred only in the presence of Cl(-). Stopped-flow measurements revealed that the HOCl-mediated MPO heme destruction was complex and occurred through transient ferric species whose formation and decay kinetics indicated it participates in heme destruction along with subsequent free iron release. MPO heme depletion was confirmed by the buildup of free iron utilizing the ferrozine assay. Hypochlorous acid, once generated, first equilibrates in the solution as a whole before binding to the heme iron and initiating heme destruction. Eliminating HOCl from the MPO milieu by scavenging HOCl, destabilizing the MPO-Compound I-Cl complex that could be formed during catalysis, and/or inhibiting MPO catalytic activity partially or completely protects MPO from HOCl insults. Collectively, this study elucidates the bidirectional relationship between MPO and HOCl, which highlights the potential role of MPO as a source of free iron.

Biological activities of the polysaccharides produced in submerged culture of two edible Pleurotus ostreatus mushrooms.

Exopolysaccharides (EPS) and internal (intracellular) polysaccharides (IPS) obtained from the Pleurotus ostreatus M2191 and PBS281009 cultivated using the batch system revealed an average of between 0.1-2 (EPS) and 0.07-1.5 g/L/day (IPS). The carbohydrate analysis revealed that the polysaccharides comprised 87-89% EPS and 68-74% IPS. The investigation of antioxidant activity in vitro revealed a good antioxidant potential, particularly for the IPS and EPS isolated from PBS281009, as proved by the EC(50) value for DPPH, ABTS scavenging activity, reducing power, and iron chelating activity.

The role of reduction in iron uptake processes in a unicellular, planktonic cyanobacterium.

In many aquatic environments the essential micronutrient iron is predominantly complexed by a heterogeneous pool of strong organic chelators. Research on iron uptake mechanisms of cyanobacteria inhabiting these environments has focused on endogenous siderophore production and internalization. However, as many cyanobacterial species do not produce siderophores, alternative Fe acquisition mechanisms must exist. Here we present a study of the iron uptake pathways in the unicellular, planktonic, non-siderophore producing strain Synechocystis sp. PCC 6803. By applying trace metal clean techniques and a chemically controlled growth medium we obtained reliable and reproducible short-term (radioactive assays) and long-term (growth experiments) iron uptake rates. We found that Synechocystis 6803 is capable of acquiring iron from exogenous ferrisiderophores (Ferrioxamine-B, FeAerobactin) and that unchelated, inorganic Fe is a highly available source of iron. Inhibition of iron uptake by the Fe(II)-specific ligand, ferrozine, indicated that reduction of both inorganic iron and ferrisiderophore complexes occurs before transport through the plasma membrane. Measurements of iron reduction rates and the inhibitory effect of ferrozine on growth supported this conclusion. The reduction-based uptake strategy is well suited for acquiring iron from multiple complexes in dilute aquatic environments and may play an important role in other cyanobacterial strains.

Heme-mediated binding of α-casein to ferritin: evidence for preferential α-casein binding to ferrous iron.

Bovine milk α-casein was identified as a ferritin-binding protein, and ferritin is known to be a heme-binding protein. In this study, we found that the binding of α-casein to bovine spleen ferritin in vitro was blocked by hemin, but not by iron-free hemin (protoporphyrin IX) or zinc-protoporphyrin IX, suggesting that the presence of iron in heme play a key role in this interaction. Indeed, the binding of α-casein to ferritin and biotinylated hemin was inhibited by adding excess ferrous ammonium sulfate (FAS). To further elucidate the binding mechanism of α-casein to biotinylated hemin, Ferrozine and nitrilotriacetic acid (NTA) were used as ferrous and ferric iron chelators, respectively. FAS-mediated inhibition of α-casein to biotinylated hemin was neutralized with Ferrozine, but not NTA, while FAS- as well as ferric chloride-mediated inhibition in their interaction was neutralized by NTA. The following ions also inhibited α-casein-biotinylated hemin binding in order of potency of inhibition: FAS (Fe(2+)) << ferric chloride (Fe(3+)) < copper sulfate (Cu(2+)) < zinc sulfate (Zn(2+)) < manganese chloride (Mn(2+)) < calcium chloride (Ca(2+)) < magnesium sulfate (Mg(2+)). These results suggests that the binding of α-casein to ferritin is heme-mediated through direct binding of α-casein to iron in the heme on the surface of ferritin molecule, and that α-casein preferentially binds Fe(2+) compared with any other metal ions, including Fe(3+).

Neurochemical alterations in lemon shark (Negaprion brevirostris) brains in association with brevetoxin exposure.

Brevetoxins are persistent, bioaccumulative, lipophilic polyether neurotoxins synthesized by Karenia brevis, a harmful algal bloom (HAB) dinoflagellate. Although some marine organisms accumulate potentially harmful levels of brevetoxins, little is known about neurotoxic effects in wild populations. Here, tissue (i.e., liver, kidney, muscle, intestine, gill, brain) brevetoxin levels (as ng PbTx-3 eq/g) and four neurochemical biomarkers (monoamine oxidase, MAO; cholinesterase, ChE; muscarinic cholinergic receptor, mAChR; N-methyl-d-aspartic acid receptor, NMDAR) were compared between eleven lemon sharks collected during a K. brevis bloom and eighteen lemon sharks not exposed to a bloom (controls) in a case-control manner. Brevetoxin levels in tissues were significantly higher in HAB-exposed sharks when compared to controls, and tissue levels (e.g., 277-3112 ng/g in livers, 429-2833 ng/g in gills) in HAB-exposed sharks were comparable to levels detected in a shark (e.g., 1223 ng/g in liver, 930 ng/g in gill) that died presumably of toxin exposure. Further, there were significant correlations between brain brevetoxin levels and ChE activity (r=-0.41; p<0.05), MAO activity (r=-0.37; p<0.05), mAChR levels (r=0.55; p<0.01), and NMDAR levels (r=-0.49; p<0.01). There were no relationships between neurochemical biomarkers and metals (total mercury, methylmercury, selenium). Overall, these results in tissues from free-ranging lemon sharks indicate that ecologically relevant exposures to brevetoxins may cause significant changes in brain neurochemistry. As disruptions to neurochemistry precede structural and functional damage to the nervous system, these results suggest that relevant exposures to HABs may be causing sub-clinical effects in lemon sharks and raise further questions about the ecological and physiological impacts of HABs on marine biota.

Effects of radio frequency magnetic fields on iron release from cage proteins.

Ferritin, the iron cage protein, contains a superparamagnetic ferrihydrite nanoparticle formed from the oxidation and absorption of Fe(2+) ions. This nanoparticle increases its internal energy when exposed to alternating magnetic fields due to magnetization lag. The energy is then dissipated to the surrounding proteic cage, affecting its functioning. In this article we show that the rates of iron chelation with ferrozine, an optical marker, are reduced by up to a factor of 3 in proteins previously exposed to radio frequency magnetic fields of 1 MHz and 30 microT for several hours. The effect is non-thermal and depends on the frequency-amplitude product of the magnetic field.

Iron-binding and anti-Fenton properties of baicalein and baicalin.

Baicalein and baicalin, the major bioactive compounds found in the Chinese herb Scutellaria baicalensis, have been shown to be effective against cancer, bacterial infections and oxidative stress diseases. However, little is known about their mechanisms of action. To probe whether iron homeostasis modulation may play a role in their bioactivity, we have investigated their iron binding characteristics under physiologically relevant conditions. A 2:1 baicalein-ferrous complex was readily formed in 20mM phosphate buffer, pH 7.2, with a binding constant approximately 2-9 x 10(11)M(-2), whereas a 1:1 baicalein-ferric complex was formed, under the same conditions, with an apparent binding constant approximately 1-3 x 10(6)M(-1). Baicalein appears to bind the ferrous ion more strongly than ferrozine, a well known iron(II) chelator. Using (1) H NMR and Zn(2+) and Ga(3+) as probes, the iron-binding site on baicalein was elucidated to be at the O6/O7 oxygen atoms of the A-ring. No binding was observed for baicalin under the same NMR conditions. Furthermore, baicalein strongly inhibits the Fe-promoted Fenton chemistry via a combination of chelation and radical scavenging mechanism while baicalin can provide only partial protection against radical damage. These results indicate that baicalein is a strong iron chelator under physiological conditions and hence may play a vital role in modulating the body's iron homeostasis. Modulation of metal homeostasis and the inhibition of Fenton chemistry may be one of the possible mechanisms for herbal medicine.

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.

Effect of different metal ions on the oxidative damage and antioxidant capacity of hyaluronic acid.

Degradation and the antioxidative effect of Na-, Zn-, Co-, Cu-, and Mn-hyaluronic acid (HA) associates were studied. Our findings revealed the protective effect of certain counterions against ROS-induced HA degradation. We could also separate the antioxidative effect of certain counterions from that of the HA by examining the effect of the counterions in their free ionic forms. The result showed that metal ions with altering oxidative status (Co(2+), Cu(2+), Mn(2+)) proved to be effective in themselves or their effect added to that of HA when HA was also effective. Moreover, the effects of Co-HA against z.rad;O(2)(-) and of Mn-HA against ONOO(-) as well as the synergic effect of Zn-HA associates where Zn(2+) is of fixed oxidative status were attributed to the structure-stabilizing complex formed between certain counterions and HA. Our examination also concerned the influence of HA associates on the indirect antioxidation related to Fe(2+) chelating. The individual effects of Zn(2+), Co(2+), and Cu(2+) were only detectable, which could be explained by the competitive displacement of ferrous from its binding site.

Membrane heme as a host factor in reducing effectiveness of dihydroartemisinin.

Plasmodium falciparum infecting alpha-thalassemic erythrocytes are resistant to artemisinin and its derivatives. Binding of the drug to hemoglobin H resulting in drug inactivation was previously demonstrated. We now show that an additional host factor, membrane heme, significantly accounted for decreased antimalarial activity of artemisinin. The antimalarial activity of dihydroartemisinin in the presence of normal and thalassemic erythrocyte membranes showed a correlation with the heme content of the membrane (r(2)=0.466, P<0.01). The correlation was more clearly seen when the drug effectiveness was correlated with the heme content of alpha-thalassemic membrane (r(2)=0.636, P<0.01). However, the drug effectiveness showed no correlation to ferrozine-reactive (free or non-heme) iron content (r(2)=0.0001, P>0.05). alpha-Thalassemic erythrocytes contained higher amounts of membrane heme (11.04+/-8.96 nmol/mg membrane protein) than those from normal and beta-thalassemia/HbE erythrocytes (2.68+/-1.28 and 3.98+/-3.98 nmol/mg membrane protein, respectively, P<0.01). Loss of drug effectiveness was also correlated with increment of heme content in membrane prepared from normal erythrocytes treated with phenylhydrazine. It is concluded that heme in both normal and thalassemic erythrocyte membranes is an important factor in drug inactivation.

Nitric oxide and peroxynitrite activate the iron regulatory protein-1 of J774A.1 macrophages by direct disassembly of the Fe-S cluster of cytoplasmic aconitase.

Posttranscriptional regulation of iron homeostasis involves, among other factors, a reversible conversion of the Fe-S enzyme cytoplasmic aconitase to a mRNA-binding iron regulatory protein (IRP-1) that lacks an Fe-S cluster. Previous studies have shown that aconitase/IRP-1 may be a target of *NO or peroxynitrite (ONOO(-)), formed after reaction of *NO with superoxide anion (O(2)(*-)); however, the mechanisms and consequences of such interactions have remained uncertain. In this study, recombinant aconitase/IRP-1 was exposed to SIN-1, whose thermal decomposition releases *NO and O(2)(*-). Results showed that SIN-1 was able to induce concomitant inactivation of aconitase and activation of IRP-1, attributable to cluster disassembly induced by ONOO(-). SIN-1 was used also in lysates of J774A.1 mouse macrophages grown under control conditions, or subjected to iron loading or starvation by treatment with hemin or desferrioxamine, respectively. Three lines of evidence confirmed that ONOO(-) activated IRP-1 by removing iron from the Fe-S cluster of cytoplasmic aconitase. First, IRP-1 activation was accompanied by iron release and loss of aconitase activity. Second, aconitase activity was recovered by reassembling Fe-S clusters with cysteine and ferrous ammonium sulfate. Third, iron release and IRP-1 activation were observed in lysates from control or iron-loaded macrophages, containing increasing levels of Fe-S clusters, but not in lysates from iron-starved macrophages, in which aconitase had already undergone cluster disassembly and switched to IRP-1. *NO was less efficient than ONOO(-) in attacking the Fe-S cluster of cytoplasmic aconitase; in fact, SIN-1-dependent iron release and IRP-1 activation were diminished by superoxide dismutase, which scavenged O(2)(*-) before it reacted with *NO to form ONOO(-). Under comparable conditions, however, both *NO and ONOO(-) inactivated an IRP-2 unable to assemble an Fe-S cluster. These results indicate that *NO and ONOO(-) may activate IRP-1 by attacking the Fe-S cluster of cytoplasmic aconitase, while also inactivating the cluster-deficient IRP-2. Such divergent actions offer clues to explain links between iron homeostasis and reactive nitrogen species in macrophages involved in inflammation or other pathophysiologic conditions.

Effects of citrinin on iron-redox cycle.

The ability of the mycotoxin citrinin to act as an inhibitor of iron-induced lipoperoxidation of biological membranes prompted us to determine whether it could act as an iron chelating agent, interfering with iron redox reactions or acting as a free radical scavenger. The addition of Fe3+ to citrinin rapidly produced a chromogen, indicating the formation of citrinin-Fe3+ complexes. An EPR study confirms that citrinin acts as a ligand of Fe3+, the complexation depending on the [Fe3+]:[citrinin] ratios. Effects of citrinin on the iron redox cycle were evaluated by oxygen consumption or the o-phenanthroline test. No effect on EDTA-Fe2+-->EDTA-Fe3+ oxidation was observed in the presence of citrinin, but the mycotoxin inhibited, in a dose-dependent manner, the oxidation of Fe2+ to Fe3+ by hydrogen peroxide. Reducing agents such as ascorbic acid and DTT reduced the Fe3+-citrinin complex, but DTT did not cause reduction of Fe3+-EDTA, indicating that the redox potentials of Fe3+-citrinin and Fe3+-EDTA are not the same. The Fe2+ formed from the reduction of Fe3+-citrinin by reducing agents was not rapidly reoxidized to Fe3+ by atmospheric oxygen. Citrinin has no radical scavenger ability as demonstrated by the absence of DPPH reduction. However, a reaction between citrinin and hydrogen peroxide was observed by UV spectrum changes of citrinin after incubation with hydrogen peroxide. It was also observed that citrinin did not induce direct or reductive mobilization of iron from ferritin. These results indicate that the protective effect on iron-induced lipid peroxidation by citrinin occurs due to the formation of a redox inactive Fe3+-citrinin complex, as well as from the reaction of citrinin and hydrogen peroxide.

NFkappaB translocation in human microvessel endothelial cells using a four-compartment subcellular protein redistribution assay.

Protein distribution profiles may be used to characterize both physiological and pathophysiological cellular changes, but rigorous biochemical assays for measuring such movements are lacking. This paper reports on a protein redistribution assay that combines reversible metal chelate-based total protein detection with a four-fraction subcellular detergent fractionation procedure. TNF-alpha stimulated cultured human omental microvessel endothelial cells are fractionated into cytosol, membrane/organelle, nuclear (envelope and associated), and cytoskeletal/DNA compartments. Protein fractions are separated electrophoretically and electroblotted or slot-blotted onto PVDF membranes without electrophoretic separation. A key feature is that total protein is measured and analyzed directly on the resultant PVDF membrane, using a Ferrozine/ferrous metal-chelate stain, without the added step of a prior solution-phase protein assay. As a result, factors that may adversely affect NFkappaB quantification, such as saturation of the solid-support membrane, are rigorously evaluated and controlled. Following removal of the Ferrozine/ferrous total protein stain, NFkappaB distribution is determined via standard immunodetection procedures. This assay reveals a new level of complexity regarding NFkappaB distribution and translocation. NFkappaB is shown to translocate from the cytosol to the membrane/organelle and cytoskeletal/DNA fractions, whereas trace levels of NFkappaB are observed in the nuclear (envelope and associated) fraction. Dose-curve analysis reveals that the response is initiated at 10 U/ml of TNF-alpha, plateaus at approximately 1000 U/ml, and remains essentially constant up to 2000 U/ml. Time-course analysis demonstrates a measurable response as early as 5 min and a peak response at approximately 30 min, after which the distribution begins to return to baseline. The assay should provide a valuable tool for rapid evaluation and mechanistic studies of NFkappaB redistribution.

Dopamine and DOPA cause release of iron from ferritin and lipid peroxidation of liposomes.

We investigated dopamine (DA)- and DOPA-related release of iron from ferritin, and lipid peroxidation of liposomes induced by the released iron. Iron release increased with increasing DA or DOPA concentrations. Effects of SOD and an oxygen-reduced environment indicated that superoxide was partly responsible for iron release. The released iron induced lipid peroxidation at relatively low concentrations of DA or DOPA, while at high concentrations, peroxidation was inhibited. These findings indicate that the risk of lipid peroxidation depends on the DA/iron or DOPA/iron ratio even if the iron concentration is low. Our findings suggest that DA-containing neurons are always at risk of oxidative damage. Furthermore, DOPA therapy may modify the nigral degeneration by reducing or accelerating ferritin iron-dependent lipid peroxidation.