Labile Iron Pool Levels Research Articles

White spot syndrome virus protein kinase 1 defeats the host cell's iron-withholding defense mechanism by interacting with host ferritin.

Iron is an essential nutrient for nearly all living organisms, including both hosts and invaders. Proteins such as ferritin regulate the iron levels in a cell, and in the event of a pathogenic invasion, the host can use an iron-withholding mechanism to restrict the availability of this essential nutrient to the invading pathogens. However, pathogens use various strategies to overcome this host defense. In this study, we demonstrated that white spot syndrome virus (WSSV) protein kinase 1 (PK1) interacted with shrimp ferritin in the yeast two-hybrid system. A pulldown assay and 27-MHz quartz crystal microbalance (QCM) analysis confirmed the interaction between PK1 and both ferritin and apoferritin. PK1 did not promote the release of iron ions from ferritin, but it prevented apoferritin from binding ferrous ions. When PK1 was overexpressed in Sf9 cells, the cellular labile iron pool (LIP) levels were elevated significantly. Immunoprecipitation and atomic absorption spectrophotometry (AAS) further showed that the number of iron ions bound by ferritin decreased significantly at 24 h post-WSSV infection. Taken together, these results suggest that PK1 prevents apoferritin from iron loading, and thus stabilizes the cellular LIP levels, and that WSSV uses this novel mechanism to counteract the host cell's iron-withholding defense mechanism. We show here that white spot syndrome virus (WSSV) ensures the availability of iron by using a previously unreported mechanism to defeat the host cell's iron-withholding defense mechanism. This defense is often implemented by ferritin, which can bind up to 4,500 iron atoms and acts to sequester free iron within the cell. WSSV's novel counterstrategy is mediated by a direct protein-protein interaction between viral protein kinase 1 (PK1) and host ferritin. PK1 interacts with both ferritin and apoferritin, suppresses apoferritin's ability to sequester free iron ions, and maintains the intracellular labile iron pool (LIP), and thus the availability of free iron is increased within cells.

Are the health effects of exercise related to changes in iron metabolism?

There are increasing number of evidences that regular exercise reduce risks of several morbidities like cancer, heart attack, type II diabetes, hypertension and slows down ageing process. The mechanism of pro-healthy effects of exercise is far from being completely understood. For example exercise increase insulin sensitivity in type II diabetics and this is related to activation of AMP kinase and increase biogenesis of mitochondria in skeletal muscle. Interestingly, increase in insulin sensitivity was also observed in patients, whose body iron stores has been reduced. The fact that exercise has been reported to influence body iron metabolism made us ponder the question whether many positive effects of exercise can be related to these changes. Iron is essential for most of cellular processes like energy metabolism, cell proliferation, synthesis of DNA and collagen, posttranslational modification of some proteins and many others. However, for our body iron like oxygen when in excess, is toxic. The elevated body iron reserves have been linked with an increased risk of health problems such as cancer, type 2 diabetes, and coronary artery disease. Total body iron accumulates when iron intake begins to exceed its loss. This data are confirmed by studies on animal models of iron-induced cancer, and diabetes. Life-style changes such as exercise, calorie restriction, a diet rich in iron chelators, and phlebotomy are all associated with the reduction of total body iron. These treatments lead to diminished risk of several morbidities. Nevertheless, studies on cell cultures demonstrated that the labile iron pool (LIP) mainly determines its toxicity rather than total amount of accumulated iron. The LIP level is regulated by several signaling pathways and gene expression. Thus, it is expected that even at a high concentration of intracellular iron, its toxicity is not obvious until a cell is able to maintain a low level of LIP. These data suggest that an effective control of body iron stores (diet, exercise, and possibly phlebotomy) would be a wise strategy for disease prevention.

Role of intracellular labile iron, ferritin, and antioxidant defence in resistance of chronically adapted Jurkat T cells to hydrogen peroxide

To examine the role of intracellular labile iron pool (LIP), ferritin (Ft), and antioxidant defence in cellular resistance to oxidative stress on chronic adaptation, a new H2O2-resistant Jurkat T cell line “HJ16” was developed by gradual adaptation of parental “J16” cells to high concentrations of H2O2. Compared to J16 cells, HJ16 cells exhibited much higher resistance to H2O2-induced oxidative damage and necrotic cell death (up to 3mM) and had enhanced antioxidant defence in the form of significantly higher intracellular glutathione and mitochondrial ferritin (FtMt) levels as well as higher glutathione-peroxidase (GPx) activity. In contrast, the level of the Ft H-subunit (FtH) in the H2O2-adapted cell line was found to be 7-fold lower than in the parental J16 cell line. While H2O2 concentrations higher than 0.1mM fully depleted the glutathione content of J16 cells, in HJ16 cells the same treatments decreased the cellular glutathione content to only half of the original value. In HJ16 cells, H2O2 concentrations higher than 0.1mM increased the level of FtMt up to 4-fold of their control values but had no effect on the FtMt levels in J16 cells. Furthermore, while the basal cytosolic level of LIP was similar in both cell lines, H2O2 treatment substantially increased the cytosolic LIP levels in J16 but not in HJ16 cells. H2O2 treatment also substantially decreased the FtH levels in J16 cells (up to 70% of the control value). In contrast in HJ16 cells, FtH levels were not affected by H2O2 treatment. These results indicate that chronic adaptation of J16 cells to high concentrations of H2O2 has provoked a series of novel and specific cellular adaptive responses that contribute to higher resistance of HJ16 cells to oxidative damage and cell death. These include increased cellular antioxidant defence in the form of higher glutathione and FtMt levels, higher GPx activity, and lower FtH levels. Further adaptive responses include the significantly reduced cellular response to oxidant-mediated glutathione depletion, FtH modulation, and labile iron release and a significant increase in FtMt levels following H2O2 treatment.

ROS-Mediated Iron Overload Injures The Hematopoiesis Of Bone Marrow By Damaging Hematopoietic Stem/Progenitor Cells In Mice

A substantial portion of patients with inherited blood disorders such as beta thalassemia, or bone marrow failure syndromes such as aplastic anemia(AA), myelodysplastic syndromes(MDS) require frequent transfusions of red blood cells. Frequent blood transfusions may lead to the excess of plasma non-transferrin -bound iron(NTBI) and iron overload occurs, which will significantly injure bone marrow (BM) function as well as induce organ dysfunctions such as liver cirrhosis, diabetes and cardiac diseases. However, the exact mechanism behind this effect remains elusive and ideal treatment needs to be explored. In our preliminary studies, we have demonstrated free iron catalyzes oxidative damage to hematopoietic cells/ mesenchymal stem cells in vitro and suppresses hematopoiesis in iron overload patients (Zhao et al.,blood, 2010 abstract; Lu et al.,blood,2012 abstract; Lu et al., Eur J Haematol, 2013). Here we observed the hematopoiesis inhibitory effects of iron overload on the basis of estabalished iron overload mice model and preliminarily disscussed the mechanism.In this study, we first established an iron overload mice model by administering different doses(12.5mg/ml,25mg/ml,50mg/ml) iron dextran by intraperitoneal injection every three days for four weeks. To confirm the efficacy of the mice model, the BM, hepatic and splenic iron deposits were observed by morphological study and the labile iron pool level (LIP) of bone marrow mononuclear cells(BMMNCs) was detected using the calcein-AM fluorescent dye. It was found that iron deposits in BM cells of iron overload mice, liver and spleen were markedly increased and the BMMNCs LIP level was much higher than that of normal control mice. The above results showed that the iron-overloaded mice model has been established successfully.Next we observed whether iron overload (25mg/ml) could affect the hematopoiesis of BM. The colony-forming cell assay was performed by culturing BMMNCs in MethoCult M3434 methylcellulose medium to evaluate hematopoietic progenitor cells(HPCs) proliferation function. The competitive repopulation assay and single-cell colony cultures of sorted hematopoietic stem cells (HSCs,CD34-Lin- sca1+c-kit+cells,LSK+)were used to validate HSCs function.The counts of BMMNCs have no significant difference. However, It was found that hematopoietic colony-forming unit (CFU-E, BFU-E, CFU-GM and CFU-mix) was much lower than that of normal control(P<0.05)(Fig.1). Notely, the number of LSK+ cells (*103/femur) was decreased significantly in iron overload mouse (26.43±3.28) compared with normal control(40.12±5.21) and the single-cell colony formation(/60wells) was reduced significantly in iron overload mouse(28.54±3.33) compared with normal control(47.93±4.82) (P<0.05). The long-term and multilineage engraftment capability of the iron-overloaded HSCs was weaken after transplantation.We then explored the possible mechanism of this inhibitory effects. Our previous studies have shown that iron overload could elevated reactive oxygen species (ROS) levels of mesenchymal stem cells and HSCs in vitro. Similarly, the intracellular ROS levels were analyzed by a flow cytometer. It was found that ROS level in iron overload BM was increased by 3.32 folds in erythroid cells, 1.51 folds in granulocytes and 4.80 folds in LSK+ cells,respectively. And also, the expression of p53, p38MAPK and p16Ink4a mRNA remained significantly elevated, which indicated that ROS related signal pathway was involved in the deficient hematopoiesis of iron overload BM.In addition, we also observed the effects of iron overload on the mice with deficient hematopoiesis exposed to 4Gy total body irradiation(TBI), which was more similar to clinical pathological conditions such as AA or MDS. It was found that BM damage caused by iron overload was aggravated in pathological conditions (primary findings were not shown).In conclusion, our study confirmed that iron overload injures the hematopoiesis of BM by enhancing oxidative stress in mice, which would be helpful to further study on the mechanism and would provide an experimental basis to find new therapeutic targets for the treatment of iron overload in patients with hematopoietic dysfunction. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Quercetin protects rat hepatocytes from oxidative damage induced by ethanol and iron by maintaining intercellular liable iron pool

Accumulating evidence has shown that ethanol-induced iron overload plays a crucial role in the development and progression of alcoholic liver disease. We designed the present study to investigate the potential protective effect of quercetin, a naturally occurring iron-chelating antioxidant on alcoholic iron overload and oxidative stress. Ethanol-incubated (100 mmol/L) rat primary hepatocytes were co-treated by quercetin (100 µmol/L) and different dose of ferric nitrilotriacetate (Fe-NTA) for 24 h. When the hepatic enzyme releases in the culture medium, redox status of hepatocytes and the intercellular labile iron pool (LIP) level were assayed. Our data showed that Fe-NTA dose dependently induced cellular leakage of aspartate transaminase and lactate dehydrogenase, glutathione depletion, superoxide dismutase inactivation, and overproduction of malondialdehyde) and reactive oxygen species (ROS) of intact and especially ethanol-incubated hepatocytes. The oxidative damage resulted from ethanol, Fe-NTA, and especially their combined treatment was substantially alleviated by quercetin, accompanying the corresponding normalization of intercellular LIP level. Iron in excess, thus, may aggravate ethanol hepatotoxicity through Fenton-active LIP, and quercetin attenuated ethanol-induced iron and oxidative stress. To maintain intercellular LIP contributes to the hepatoprotective effect of quercetin besides its direct ROS-quenching activity.

Effect and mechanism of iron-catalyzed oxidative stress on mesenchymal stem cells

To explore effect of iron overload on the proliferation and apoptosis of mesenchymal stem cell(MSCs) and the possible mechanism. Iron overload model of MSCs was established by adding ferric ammonium citrae (FAC) into the culture medium at different concentrations (100, 200, 400 Μmol/L) and incubated for different lengths of time (12, 24, 48 h). The levels of labile iron pool (LIP) and reactive oxygen species (ROS) were measured to confirm oxidative stress state in the model. Changes in cell proliferation and apoptosis after iron overload were measured through population double time(DT)and annexin V-PI assay. Finally, the expressions of phosphorylated p38 mitogen activated protein kinase (P-p38MAPK), p38MAPK, protein kinase B (AKT), and p53 were determined through Western blot analysis to investigate which ROS-mediated signaling pathway was involved in this process. The LIP level of MSCs was significantly increased by FAC treatment at 400 Μmol/L (mean fluorescence intensity 482.49±20.96 vs. 303.88±23.37, P<0.05). The level of intracellular ROS was positively correlated with the concentration of FAC and reached a peak level when cultured with 400 Μmol/L FAC (P<0.05).After treatment with 400 Μmol/L FAC at different time points (12 h, 24 h, and 48 h), the DT of MSCs was (1.47± 0.11) d, (1.80±0.13) d, and (2.04±0.14) d, respectively, which was signifcantly longer than that of the control, which was(1.20±0.05)d (P<0.05).The apoptosis rate was also significantly higher in iron overload group[(3.51±1.17)% vs.(0.66±0.62)%, P<0.05]with consequent increase in the expressions of P-p38MAPK, p38MAPK, and p53 proteins in iron overload group, while no significant difference was found in the expression of AKT. Iron overload can inhibit the proliferation of MSCs and induce their apoptosis through the generation of ROS, which is probably due to the stimulation of p38MAPK- p53 signaling pathway.

Combination Treatments of 1-(N-Acetyl-6-Aminohexyl)-3-Hydroxy-2- Methylpyridin-4-One (CM1) With Deferiprone and Desferrioxamine Reduced Labile Iron Pool and Protected Oxidative Stress in Iron-Loaded Cultured Hepatocytes

Iron overload associated with oxidative stress is a serious problem in transfusion-dependent patients with β-thalassemia major. The increased iron overload in several organs may be caused by higher intestinal absorption along with less intensive chelation therapy. Liver iron overload could in turn facilitate the development or persistence of chronic progressive liver disease. Previous studies have shown that chelation with desferrioxamine (DFO) and deferiprone (DFP) substantially reduced body-iron scores in β-thalassemia patients with transfusional iron overload. We have synthesized and characterized a new bidentate iron chelator, 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2- methylpyridin-4-one (CM1). The compound can efficiently scavenge iron from both ferrous and ferric salts and plasma non-transferrin bound iron (NTBI). In this study we have studied the efficacy of the CM1 treatment on the decrease of levels of the labile iron pool (LIP) and reactive oxygen species (ROS) in iron-loaded mouse hepatocyte and HepG2 cell cultures. The isolated hepatocytes were treated with DFP, DFO and CM1 at different concentrations. The treated cells were analyzed for intracellular LIP using the calcein fluorescent technique and ROS levels using the dichlorofluorescein (DCF) fluorescent method. It was found that CM1 reduced the levels of intracellular LIP and hydrogen peroxideinduced ROS in both treated cells in a concentration-dependent manner. The combination treatment of CM1 with 25 μM DFP and DFO was demonstrated to decrease the levels of the LIP in both cells and tended to reduce the levels of ROS in HepG2 cells. Our findings support the evidence of iron-chelating and free radical-scavenging activities of CM1 in the livers with iron overload, which potentially can protect against oxidative liver inflammation and fibrosis. The efficacy of the CM1 treatment needs to be further investigated intensively under in vivo conditions.

Green tea extract and epigallocatechin 3-gallate reduced labile iron pool and protected oxidative stress in iron-loaded cultured hepatocytes

Cellular and mitochondrial damage can be caused by labile iron pool (LIP) and mediated by reactive oxygen species (ROS). Livers of the thalassemias have highly increased levels of LIP and ROS. Green tea extract (GTE) and epigallocatechin 3-gallatte (EGCG) can potentially protect liver inflammation, fibrosis and cancer due to their anti-oxidative and iron-chelating activities. We studied the effects of GTE and EGCG on intracellular LIP and ROS, and mitochondrial membrane potential (ΔΨm) in mouse hepatocyte and HepG2 cell cultures using specific fluorescent techniques. Treatment with GTE (12.5 - 25 mg/dl) and EGCG (25 - 50 μM) significantly lowered levels of ΔΨm in the mouse hepatocytes; however, combined treatment of 25 μM DFP with GTE and EGCG did not enhance the decrease of hepatic ΔΨm. The results showed that GTE and EGCG effectively removed the intracellular LIP and ROS, and relieved the mitochondria membrane collapse of the liver cells, suggesting a hepatoprotective effect of green tea extract and EGCG in the hepatocytes with iron overload. Their actions might be related to iron-chelating and free radical-scavenging capacities. Whether the effects can improve iron overload and oxidative stress in thalassemia patients remains to be seen upon further examination.

Effects of oxidative stress on hematopoiesis of hematopoietic stem and progenitor cells with iron overload

To establish a model of hematopoietic stem and progenitor cells with iron overload derived from umbilical cord blood (UCB) cells and explore the effects of reactive oxygen species (ROS) on the hematopoiesis of hematopoietic stem and progenitor cells with iron overload. The model was established by adding different concentrations (50, 100, 200, 400 µmol/L)of ferric citrate (FAC) into mononuclear cells from UCB and culturing for different times (6, 12, 24 h). The UCB cells were divided into 4 groups: control group, group FAC, group FAC+N-acetyl-L-cysteine (NAC) and group FAC+ L-Glutathione (GSH). Then the changes of ROS, labile iron pool (LIP), apoptosis, the capacity of hematopoietic colony forming (CFU-E, BFU-E, CFU-GM, CFU-mix) and the percentage and the numbers of CD34(+), CD33(+), GlyA(+) cells were detected. And the changes of these indices were tested after the treatment of iron overload UCB with antioxidants (NAC and GSH). UCB cells were cultured with the addition of FAC at different concentrations for different times. The level of total ROS increased in time and concentration-dependent manners. The intracellular level of ROS peaked when cultured at 200 µmol/L of FAC for 24 hours. Cells were treated with antioxidants NAC or GSH after cultured with 200 µmol/L FAC for 24 hours. Then the ROS levels of total cells, myeloid cells and erythroid cells decreased markedly versus normal controls. The LIP of total cells, myeloid cells and erythroid cells increased markedly when cells were cultured at 200 µmol/L of FAC for 24 hours versus normal controls (P < 0.05). NAC and GSH had no effect on the level of LIP. The apoptotic rates of FAC-treated cells [(20.90 ± 3.45)%] increased significantly versus normal controls [(9.20 ± 1.29)%] (P < 0.05). The capacity of hematopoietic colony forming in FAC treated cells decreased markedly versus normal controls. The percentage and numbers of CD34(+), CD33(+), GlyA(+) cells of FAC-treated cells also decreased significantly versus normal controls (P < 0.05). And these changes could be recovered by the addition of NAC or GSH. Oxidative stress plays an important role in the injuries of hematopoiesis of hematopoietic stem and progenitor cells with iron overload by inducing the generation of ROS. These findings may help us find a specific target and improve the therapeutic efficacy of ineffective hematopoiesis in patients with iron overload.

Effects of the iron‐chelating agent deferoxamine on triethylene glycol dimethacrylate, 2‐hydroxylethyl methacrylate, hydrogen peroxide‐induced cytotoxicity

Triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxylethyl methacrylate (HEMA) are known to deplete glutathione in mammalian cells, generate reactive oxygen species (ROS), and cause oxidative stress. In this study, we investigated whether hydroxyl radicals (·OH), the most lethal and genotoxic ROS, and the Fenton reaction are involved in the cytotoxicity of resin monomers to four different cell types, namely MC3T3-E1 preosteoblasts, human dental pulp cells (HDPCs), human gingival fibroblasts, and L929 mouse fibroblasts. Deferoxamine (DFO), an iron chelating agent, effectively protected MC3T3-E1 cells from resin monomer-induced cytotoxicity, indicating that cytotoxicity was caused primarily by hydroxyl radicals. However, DFO only had a protective effect against relatively high concentrations of TEGDMA and HEMA in HDPCs and human gingival fibroblasts, and resin monomer-induced cytotoxicity in L929 was not attenuated by DFO. A labile iron pool (LIP) was detectable only in MC3T3-E1 cells among the four cell types. This indicates that the generation of hydroxyl radicals induced by resin monomers is likely dependent on LIP levels. In contrast to resin monomers, hydrogen peroxide (H(2)O(2))-induced cytotoxicity was not prevented by DFO in any of the cell types examined, although hydroxyl radicals were detected in MC3T3-E1 cells and HDPCs on exposure to exogenous H(2)O(2). This result suggests that generation of hydroxyl radicals is not always the primary cause of cytotoxicity in H(2)O(2)-treated cells.

Nitric Oxide Suppresses Tumor Cell Migration Through N-Myc Downstream Regulated Gene-1 (NDRG1) Expression: the Role of Chelatable Iron

Background: The cytosolic labile iron pool (LIP) is a transitory, catalytically active compartment that has been implicated in cell iron homeostasis and in metal-induced cytotoxicity.Aims: We attempted to define LIP levels in living hepatocytes derived from chronic overloaded rats and from normal hepatocytes either acutely loaded with iron or depleted by chelation.Methods: LIP levels were measured in living rat hepatocytes derived from normal and iron-fed rats.Results: Steady-state LIP levels in untreated hepatocytes (∼0.2 μM) were raised by 1.8-fold following iron loading and were reduced by 0.66-fold by short-term chelation treatment. Changes in LIP were accompanied by the corresponding changes in iron-responsive protein (IRP) activity and ferritin levels, that, in rat hepatocytes isolated from chronically loaded animals, raised by ∼19-fold.Conclusions: Whereas ferritin levels provide an index of long-term or cumulative iron loading, LIP measurements provide an “instantaneous” parameter of iron availability within hepatocytes. The latter was associated with the cell chelatable pool in cells derived from normal and iron-loaded animals, both of which showed similar accessibility to iron chelators.

Chronic expression of H-ferritin in dopaminergic midbrain neurons results in an age-related expansion of the labile iron pool and subsequent neurodegeneration: implications for Parkinson's disease

While ferritin elevation within dopaminergic (DA) neurons of the substantia nigra (SN) is protective against neurodegeneration elicited by two toxin models of Parkinson's disease (PD), MPTP and paraquat, in young animals, its prolonged elevation results in a selective age-related neurodegeneration. A similar age-related neurodegeneration has been reported in iron regulatory protein 2-deficient (IRP2 −/−) mice coinciding with increased ferritin levels within degenerating neurons. This has been speculated to be due to subsequent reductions in the labile iron pool (LIP) needed for the synthesis of iron–sulfur-containing enzymes. In order to assess whether LIP reduction is responsible for age-related neurodegeneration in our ferritin transgenics, we examined LIP levels in ferritin-expressing transgenics with increasing age. While LIP levels were reduced within DA SN nerve terminals isolated from young ferritin transgenics compared to wildtype littermate controls, they were found to be increased in older transgenic animals at the age at which selective neurodegeneration is first noted. Furthermore, administration of the bioavailable iron chelator, clioquinol (CQ), to older mice was found to protect against both increased LIP and subsequent dopaminergic neurodegeneration. This suggests that age-related neurodegeneration in these mice is likely due to increased iron availability rather than its reduction. This may have important implications for PD and other related neurodegenerative conditions in which iron and ferritin have been implicated.

Ferritin Heavy Chain–Mediated Iron Homeostasis and Subsequent Increased Reactive Oxygen Species Production Are Essential for Epithelial-Mesenchymal Transition

The epithelial-mesenchymal transition (EMT) plays a critical role in tumor progression. To obtain a broad view of the molecules involved in EMT, we carried out a comparative proteomic analysis of transforming growth factor-beta1 (TGF-beta1)-induced EMT in AML-12 murine hepatocytes. A total of 36 proteins with significant alterations in abundance were identified. Among these proteins, ferritin heavy chain (FHC), a cellular iron storage protein, was characterized as a novel modulator in TGF-beta1-induced EMT. In response to TGF-beta1, there was a dramatic decrease in the FHC levels, which caused iron release from FHC and, therefore, increased the intracellular labile iron pool (LIP). Abolishing the increase in LIP blocked TGF-beta1-induced EMT. In addition, increased LIP levels promoted the production of reactive oxygen species (ROS), which in turn activated p38 mitogen-activated protein kinase. The elimination of ROS inhibited EMT, whereas H2O2 treatment rescued TGF-beta1-induced EMT in cells in which the LIP increase was abrogated. Overexpression of exogenous FHC attenuated the increases in LIP and ROS production, leading to a suppression of EMT. We also showed that TGF-beta1-mediated down-regulation of FHC occurs via 3' untranslated region-dependent repression of the translation of FHC mRNA. Moreover, we found that FHC down-regulation is an event that occurs between the early and highly invasive advanced stages in esophageal adenocarcinoma and that depletion of LIP or ROS suppresses the migration of tumor cells. Our data show that cellular iron homeostasis regulated by FHC plays a critical role in TGF-beta1-induced EMT.

Signal transduction pathways are altered by cellular labile iron

The cellular labile iron pool (LIP), which represents &lt;5% of the total cellular iron is associated with a diverse population of ligands such as organic anions, polypeptides, and surface components of membranes. LIP as a self‐regulatory pool that is sensed by cytosolic iron regulatory proteins (IRPs) and feedback regulated by IRP‐dependent expression of iron import and storage proteins. Variation in the LIP can also contribute to basic cellular signaling events. In these studies we examined the effect of LIP variation on cell surface processes mediated by protein kinase C (PKC) in a model system of neuroblastoma cells. Levels of isoforms of PKC were highly sensitive to LIP while PKC beta isoform being more susceptible to change. Confocal microscopy studies suggest increased ubiqitination and internalization of membrane proteins and are consistent with qRT‐PCR gene array analysis of signaling pathways. Lower LIP levels result in increased ubiqitination, increased phosphorylation, and lower rates of endocytic protein recycling. The proximal "switch" for this activation is being explored. This ongoing study is supported by USDA (# 2007‐35200‐18231).

Flow cytometry measurement of the labile iron pool in human hematopoietic cells

Iron is important for many biological processes, and its deficiency or excess is involved in pathological conditions. Although most iron is firmly bound (e.g., in hemoglobin), some, the labile iron pool (LIP), is bound to low-affinity ligands. The level of LIP is regulated to meet the cell's requirements for iron but prevent excess. We describe herein a multiparameter flow cytometry procedure for measuring LIP in various human hematopoietic cells. Peripheral blood and bone marrow (BM) cells were loaded with calcein-AM, washed, and then incubated with or without the high-affinity iron-chelator Deferiprone (L1). Specific cell subpopulations were identified based on side-light scattering and expression of surface antigens. LIP was determined based on the ability of L1 to bind and remove iron from calcein and thereby increase the fluorescence emitted by the cells. Blood cells differ in their LIP content in the order monocytes > PMN > RBC > lymphocytes. Analysis of BM cells indicated a similar tendency among precursors of the different lineages. The results also showed that among myeloid precursors, LIP increases along cell maturation. Flow cytometry might be useful for evaluating LIP in various diseases and for studying the efficacy of iron-chelators.

Sulfide increases labile iron pool in RD4 cells

A linkage between sulfur and iron metabolism has been suggested since sulfide has the ability to release iron from ferritin in the presence of iron acceptors in vitro. Nevertheless, this linkage is still lacking evidence in vivo as well as in cellular models. In this study we have treated human RD4 skeletal muscle cells with sodium sulfide and measured the level of the labile iron pool (LIP) as well as the intracellular sulfide concentration. We have also detected the amounts of L-ferritin protein as well as the iron regulatory protein 2 (IRP2). The sulfide treatment resulted in a 100% increase in the amount of LIP after 1 and 2 h. We also found that the raise of the LIP levels was coupled to an elevation of the amounts of intracellular sulfide that increased by 60%. The bioavailability of the released iron was confirmed by a 100% increase in L-ferritin protein as well as a 60% decrease of the IRP2 protein levels. These results suggest that there is a linkage between sulfur metabolism and intracellular iron regulation in mammalian cells.

Does the cellular labile iron pool participate in the oxidation of 2′,7′-dichlorodihydrofluorescein?

The fluorogenic probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) is widely used for the estimation of oxidative stress in cells. It is known that 2′,7′-dichlorodihydrofluorescein (H2DCF), product of intracellular hydrolysis of H2DCF-DA, is oxidized to the fluorescent compound, DCF, mainly by hydrogen peroxide (H2O2) in the presence of catalysts. The present study was aimed at answering the question whether the labile iron pool (LIP) may contribute to the oxidation of H2DCF in cellular systems. The membrane-permeable lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) was found to inhibit oxidation of the probe by H2O2 dependent on ferrous ions but not by peroxidase or superoxide dismutase in defined in vitro systems. When applied to cells, the probe inhibited considerably oxidation of H2DCF in V79 Chinese hamster fibroblasts and two murine lymphoma L5178Y(LY) sublines (LY-R, LY-S) differing in LIP level, the extent of inhibition being greater in the LY-R line of higher LIP level. These results demonstrate that LIP is a significant factor determining the rate of intracellular H2DCF oxidation.

Distinct Roles of Basal Steady-State and Induced H-Ferritin in Tumor Necrosis Factor-Induced Death in L929 Cells

Tumor necrosis factor (TNF) alpha is a cytokine capable of inducing caspase-dependent (apoptotic) cell death in some cells and caspase-independent (necrosis-like) cell death in others. Here, using a mutagenesis screen for genes critical in TNF-induced death in L929 cells, we have found that H-ferritin deficiency is responsible for TNF resistance in a mutant line and that, upon treatment with TNF, this line fails to elevate levels of labile iron pool (LIP), critical for TNF-induced reactive oxygen species (ROS) production and ROS-dependent cell death. Since we found that TNF-induced LIP in L929 cells is primarily furnished by intracellular storage iron, the lesser induction of LIP in H-ferritin-deficient cells results from a reduction of intracellular iron storage caused by less H-ferritin. Different from some other cell lines, the H-ferritin gene in L929 cells is not TNF inducible; however, when H-ferritin is expressed in L929 cells under a TNF-inducible system, the TNF-induced LIP and subsequent ROS production and cell death were all prevented. Thus, LIP is a common denominator of ferritin both in the enhancement of cell death by basal steady-state H-ferritin and in protection against cell death by induced H-ferritin, thereby acting as a key determinant of TNF-induced cell death.

Increased iron-induced oxidative stress and toxicity in scrapie-infected neuroblastoma cells

The mechanisms behind the pathology of prion diseases are still unknown, but accumulating evidence suggests oxidative impairment along with metal imbalances in scrapie-infected brains. In this study, we have investigated iron-induced oxidative stress in scrapie-infected mouse neuroblastoma N2a (ScN2a) cells. Uninfected N2a and ScN2a cells were treated with ferric ammonium citrate (FAC) for 1–16 h, and the levels of labile iron pool (LIP), the formation of reactive oxygen species (ROS), cell viability and ferritin protein levels were measured. The increase in LIP in N2a cells was transient with a quick recovery to normal levels within 4 h accompanied by a moderate increase of formation of ROS after 3 h followed by the decrease to the basal level. In ScN2a cells, the increase in LIP was lower, but the process of recovery was prolonged and accompanied by high ROS formation and decreased cell viability. Ferritin protein levels were significantly lower in ScN2a cells than in wild-type cells in all iron treatments. These results suggest that ScN2a cells are more sensitive to iron treatment as compared to wild-type cells with respect to ROS formation and cell viability, and that ferritin deficiency in infected cells may contribute to iron-induced oxidative stress in scrapie-infected cells.

Induction of iron regulatory protein 1 RNA-binding activity by nitric oxide is associated with a concomitant increase in the labile iron pool: implications for DNA damage

Iron regulatory protein 1 (IRP1) is a bifunctional [4Fe–4S] protein that controls iron homeostasis. Switching off its function from an aconitase to an apo-IRP1 interacting with iron-responsive element-containing mRNAs depends on the reduced availability of iron in labile iron pool (LIP). Although the modulation of IRP1 by nitric oxide has been characterized, its impact on LIP remains unknown. Here, we show that inhibition of IRP1 aconitase activity and induction of its IRE-binding activity during exposure of L5178Y mouse lymphoma cells to NO are associated with an increase in LIP levels. Removal of NO resulted in a reverse regulation of IRP1 activities accompanied by a decrease of LIP. The increased iron burden in LIP caused by NO exacerbated hydrogen peroxide-induced genotoxicity in L5178Y cells. We demonstrate that the increase in LIP levels in response to chronic but not burst exposure of L5178Y cells to NO is associated with alterations in the expression of proteins involved in iron metabolism.