Iron Uptake In Cells Research Articles

Oh, NO! The Ironic Route to Neurotoxicity

Cheah et al. investigated the role of Dexras1, a Ras-family guanosine triphosphatase, and uncovered a pathway linking activation of the N -methyl-D-aspartate-type glutamate receptor (NMDAR) to regulation of iron homeostasis and implicating changes in iron uptake in NMDAR-mediated neurotoxicity. Both neuronal nitric oxide synthase (nNOS) and Dexras1 bind to the adaptor protein CAPON; NMDAR stimulation leads to the calcium-dependent activation of nNOS, enabling the S -nitrosylation and activation of Dexras1. The consequences of Dexras1 activation, however, have been unclear. Cheah et al. identified peripheral benzodiazepine receptor (PBR)-associated protein (PAP7) as a potential Dexras1 binding partner through a screen of a rat whole-brain cDNA library. The relationship was confirmed with pulldown assays using human embryonic kidney (HEK) 293 cells (which have little endogenous PAP7 and Dexras1) transfected with glutathione S -transferase-PAP7 and Myc-tagged Dexras1. PAP7 bound the iron importer divalent metal transporter (DMT1), and coimmunoprecipitation analysis revealed that Dexras1, PAP7, and DMT1 existed as a ternary complex in mouse brain. Overexpression of Dexras1 in HEK293 cells enhanced iron uptake, an effect that was markedly enhanced by cotransfection with PAP7. Exposure of cells to nitric oxide (NO) donors elicited S -nitrosylation of Dexras1 and stimulated Dexras1-dependent iron uptake in undifferentiated PC12 cells (which have abundant endogenous PAP7 and Dexras1). Furthermore, NMDA stimulated iron uptake in primary cortical neurons from wild-type but not nNOS knockout mice. Intriguingly, a cell-permeable iron chelator inhibited an NMDA-dependent increase in the formation of reactive oxygen species as well as NMDA-mediated neurotoxicity. Thus, the authors conclude that NMDA stimulates NO-dependent increase in iron uptake, which seems to play a role in NMDA-dependent neuronal death. J. H. Cheah, S. F. Kim, L. D. Hester, K. W. Clancy, S. E. Patterson III, V. Papadopoulos, S. H. Snyder, NMDA receptor-nitric oxide transmission mediates neuronal iron homeostasis via the GTPase Dexras1. Neuron 51 , 431-440 (2006). [PubMed]

Apical distribution of HFE–β2-microglobulin is associated with inhibition of apical iron uptake in intestinal epithelia cells

Mutations in the HFE gene result in hereditary hemochromatosis, a disorder of iron metabolism characterized by increased intestinal iron absorption. Based on the observation that ectopic expression of HFE strongly inhibits apical iron uptake (Arredondo et al., 2001, FASEB J 15, 1276-1278), a negative regulation of HFE on the apical membrane transporter DMT1 was proposed as a mechanism by which HFE regulates iron absorption. To test this hypothesis, we investigated: (i) the effect of HFE antisense oligonucleotides on apical iron uptake by polarized Caco-2 cells; (ii) the apical/basolateral membrane distribution of HFE, beta-2 microglobulin and DMT1; (iii) the putative molecular association between HFE and DMT1. We found that HFE antisense treatment reduced HFE expression and increased apical iron uptake, whereas transfection with wild-type HFE inhibited iron uptake. Thus, an inverse relationship was established between HFE levels and apical iron uptake activity. Selective apical or basolateral biotinylation indicated preferential localization of DMT1 to the apical membrane and of HFE and beta-2 microglobulin (beta2m) to the basolateral membrane. Ectopic expression of HFE resulted in increased distribution of HFE-beta2m to the apical membrane. The amount of HFE-beta2m in the apical membrane inversely correlated with apical iron uptake rates. Immunoprecipitations of HFE or beta2m with specific antibodies resulted in the co-precipitation of DMT1. These results sustain a model by which direct interaction between DMT1 and HFE-beta2m in the apical membrane of Caco-2 cells result in down-regulation of apical iron uptake activity.

P3-366: The complexation of amyloid-β with heme forms a peroxidase: Mechanism of neurotoxicity in Alzheimer’s disease

The molecular mechanism by which Amyloid–β (Aβ) peptide causes the neurodegeneration in Alzheimer's disease (AD) is not known. We previously proposed that excessive Aβ binds to regulatory heme, triggering functional heme deficiency (HD), causing the key cytopathologies of AD including mitochondrial dysfunction, loss of complex IV, iron accumulation, and oxidative stress (Atamna et al. 2004, PNAS). In the current study we searched for additional evidence that Ab binding to regulatory heme is the mechanism by which Aβ causes HD. The experiments included techniques of tissue culture, biochemistry, radiobiology, and HPLC. We found that heme binds to Aβ forming an Aβ–heme complex. We also found that Aβ induces heme synthesis and iron uptake in human neuroblastoma cells, which provides strong support that Aβ triggers HD. Interestingly, we also found that the Aβ–heme complex is a peroxidase, which catalyzes the oxidation of serotonin and DOPA by H2O2. Curcumin, which lowers oxidative damage in the brain in a mouse model for AD, inhibits the Aβ–heme peroxidase. The binding of Aβ to heme supports a unifying mechanism by which elevated levels of Aβ induces HD, causes oxidative damage to macromolecules, and depletes specific neurotransmitters. The relevance of the depletion of regulatory heme by excessive Aβ and Aβ–heme peroxidase to mitochondrial dysfunction, oxidative stress, neurotoxicity and other cytopathologies of AD will be discussed.

Overexpression of the zinc transporter Zip14 increases non‐transferrin‐bound iron uptake in cells

Zip14 is a member of the SLC39A zinc transporter family, which is involved in zinc uptake by cells. Furthermore, up-regulation of Zip14 by IL-6 likely contributes to the hepatic zinc accumulation and hypozincemia of inflammation. At least one member of the SLC39A family, IRT1 from Arabidopsis thaliana, is a metal transporter that can transport other trace elements, such as iron and manganese, in addition to zinc. The objective of this study was to analyze the capability of Zip14 to mediate non-transferrin-bound iron uptake by transiently transfecting HEK293H cells with mouse Zip14 cDNA. This transfection increased 65Zn uptake by many fold compared to a transfection with vector alone. Addition of ferric citrate inhibited Zip14-mediated 65Zn uptake in the transfected cells, suggesting that iron competes with zinc for this transporter. After transfection, 59Fe uptake was also assayed by incubating the cells at pH 7.0 with 59Fe-labeled ferric citrate. Thereafter, cells were treated with the proteolytic enzyme Pronase to remove plasma membrane-bound iron. Zip14-overexpressing cells internalized more 59Fe over time (P < 0.05) than did cells transfected with empty vector. After 60 min of incubation, this difference was close to four fold. The addition of a 10-fold molar excess of the ferrous chelator bathophenanthroline inhibited 90% of Zip14-mediated iron uptake. Collectively, these results indicate that Zip14 can mediate the uptake of non-transferrin-bound ferrous iron by cells at physiologic pH. Since Zip14 is very highly expressed in the liver compared to other tissues, this member of the SLC39A transporter family could be involved in hepatic iron accumulation during iron overload conditions such as hemochromatosis. Supported by NIH grants DK065064 and DK31127.

Activation of Transferrin Receptor 1 by c-Myc Enhances Cellular Proliferation and Tumorigenesis

Overexpression of transferrin receptor 1 (TFRC1), a major mediator of iron uptake in mammalian cells, is a common feature of human malignancies. Therapeutic strategies designed to interfere with tumor iron metabolism have targeted TFRC1. The c-Myc oncogenic transcription factor stimulates proliferation and growth by activating thousands of target genes. Here we demonstrate that TFRC1 is a critical downstream target of c-Myc. Using in vitro and in vivo models of B-cell lymphoma, we show that TFRC1 expression is activated by c-Myc. Chromatin immunoprecipitation experiments reveal that c-Myc directly binds a conserved region of TFRC1. In light of these findings, we sought to determine whether TFRC1 is required for c-Myc-mediated cellular proliferation and cell size control. TFRC1 inhibition decreases cellular proliferation and results in G1 arrest without affecting cell size. Consistent with these findings, expression profiling reveals that TFRC1 depletion alters expression of genes that regulate the cell cycle. Furthermore, enforced TFRC1 expression confers a growth advantage to cells and significantly enhances the rate of c-Myc-mediated tumor formation in vivo. These findings provide a molecular basis for increased TFRC1 expression in human tumors, illuminate the role of TFRC1 in the c-Myc target gene network, and support strategies that target TFRC1 for cancer therapy.

Effects of Green and Black Tea on Iron Uptake, Storage, and Availability for Free Radical Reactions

The interaction of iron with tea is important to health not only because tea polyphenols bind iron, but also because polyphenols may release iron from ferritin storage, resulting in free radical formation. In this study, we tested the effects of green and black teas on iron uptake and ferritin storage in Caco-2 cells, and on iron availability for free radical reactions in a cell-free system. Green and black teas (3 types each) were brewed at 1g/100ml for 5 minutes and tested for total phenol content. The two green and black teas most similar in total phenol content were used for subsequent experiments. Caco-2 cells were grown to confluence over 21 days and subsequently treated for 24 hours with 1, 2, or 4% green or black teas in the presence or absence of 100uM ferrous sulfate and 1mM vitamin C. Green tea (at 2 and 4%) and black tea (at 4% only) reduced soluble intracellular iron content, apical iron content (at all levels except 1% green), as well as intracellular ferritin (green at 2 and 4%, black at 4%) in Caco-2 cells (P < 0.05). Both black and green teas, at 15% strength, released iron from ferritin storage and induced significant (p < 0.0001) increases in hydroxyl radical formation in the presence of 110mg/ml ferritin. These data indicate that green and black teas reduce soluble intracellular and apical iron levels, and that they also reduce intracellular ferritin levels. Green tea was most effective at reducing intracellular iron levels. In addition, both teas are capable of releasing iron from ferritin storage and inducing free radical formation in the presence of ferritin. This project was supported by USDA.

A comparison of methods for estimating the bioavailability of elemental iron food fortificants

We evaluated the physical properties (particle size, surface area, Fisher subsieve size, apparent and pycnometric density) and bioavailability based on five screening procedures (dissolution rate in 0.1 mol/L HCl, relative biological value with respect to ferrous sulfate by the AOAC rat hemoglobin repletion method (RBV), dialyzability and Caco-2 cell iron uptake both after simulated in vitro digestion, and plasma iron tolerance in human volunteers) of nine commercial elemental iron powder food fortificants. Particle size distribution, surface area, Fisher subsieve size, dissolution rate and RBV were significantly correlated and consistent for powders of the same production type, but varied significantly between powder types. Carbonyl and electrolytic iron powders had the highest predicted bioavailability values; results for the reduced iron products were lower and variable. The predictive values of dissolution rate and RBV were confirmed for electrolytic and hydrogen reduced iron in a six month human efficacy trial; relative absorption values, compared with ferrous sulfate, were 77% and 49% in the human trial. The comparable values for dissolution rate and RBV were 73% and 38%, and 54% and 42% respectively. Dialysis, Caco-2 cell iron uptake and plasma iron tolerance tests did not provide data that would have improved the precision of predicted bioavailability estimates. We conclude that the bioavailiability of commecial iron powders used for food fortification varies significantly and that the dissolution rate in HCl under standardized conditions is the most practical and cost effective method for initial screening for bioavailability.

Cassava (Manihot esculenta) has high potential for iron biofortification

Since cassava is a crop targeted for biofortification, we studied the relationship between iron content and iron bioavailability in three cassava varieties. Phenolic compounds, ascorbic acid, carotenoids, valence of iron and myo-inositol hexakisphosphate (IP6) were also measured. The in vitro digestion/Caco-2 model was used to assess bioavailable iron. Cassava is used in several traditional dishes that are represented by two preparations; cooked (e.g. soups, to be fried, or to be covered with salsa) and bread flour (e.g. dried and grinded for further used as an ingredient in several bread making recipes). More Fe was present in the bread flour (10.5-18.3 mg g-1) than in the cooked samples (5.6–7.5 mg g-1). Iron solubility ranged from 6–36 % in the cooked samples and from 17–30% in the bread flour samples. No phenolic compounds or ascorbic acid were detected. IP6 concentration was 2.0-4.9-mmol g-1. With respect to carotenoids only all-trans-b-carotene was found both in the cooked and flour samples at concentrations of 0.14-0.44-ng g-1. Caco-2 cell iron uptake of 0.082 mmoles of intrinsic cassava Fe was comparable to the uptake from 1mmole of FeCl3, indicating that cassava Fe was highly available within both the cooked and bread flour samples. This relatively high level of availability appears to be due to enhanced levels of Fe+2, suggesting the presence of a reducing compound in the cassava. These results suggest that Fe from cassava may be highly available, and that the cassava matrix may enhance the bioavailability of Fe of other sources of Fe consumed in the same meal. Funding: HarvestPlus and USDA-ARS.

Inhibition of iron and copper uptake by iron, copper and zinc

Interactions of micronutrients can affect absorption and bioavailability of other nutrients by a number of mechanisms. In aqueous solutions, and at higher uptake levels, competition between elements with similar chemical characteristics and uptake process can take place. The consequences of these interactions may depend on the relative concentrations of the nutrients. In this work, we measure the effects of increasing concentrations of iron, zinc, and copper on iron and copper uptake in Caco-2 cells. Intracellular Fe or Cu levels were affected by incubating with increased concentrations of metals. However, when the cells already had different intracellular metal concentration, the uptake of Fe or Cu was nor affected. In competition studies, we showed that Cu and Zn inhibited Fe uptake, and while Fe inhibited Cu uptake, Zn did not. When the three metals were given together (1:1:1 ratio), Fe or Cu uptake was inhibited approximately 40%. These results point to a potential risk in the absorption and bioavailability of these minerals by the presence of other minerals in the diet. This aspect must be considered in food supplementation and fortification programs.

Characterization of mitochondrial iron uptake in HepG2 cells

There is increasing evidence that accumulation of redox-active iron in mitochondria leads to oxidative damage and contributes to various neurodegenerative diseases, such as Friedreich's ataxia and Parkinsons disease. In this work, we examined the existence of regulatory mechanisms for mitochondrial iron uptake and storage. To that end, we used rhodamine B-[(1,10-phenanthrolin-5-yl)amino carbonyl] benzyl ester, a new fluorescent iron-sensitive probe that is targeted specifically to the mitochondrion. We found that extracellular iron was incorporated readily into mitochondria in an apparently saturable process. Moreover, the rate of iron incorporation responded to the Fe status of the cell, an indication that the mitochondrion actively regulates its iron content.

Effect of aging time on the availability of freshly precipitated ferric hydroxide to coastal marine diatoms

Cell growth and iron uptake of the coastal marine diatoms Chaetoceros sociale and Thalassiosira weissflogii were studied in the presence of short-aged amorphous ferric hydroxide (am-Fe(III)) media. These were prepared by aging for 1 day, 3 days, and 3 weeks after adding a small amount of ferric iron acidic stock solution to autoclaved filtered seawater and were experimentally measured in culture experiments at 10°C for C. sociale and 20°C for T. weissflogii. The order of cell yields for both species was: 1-day aged am-Fe(III) >3-day aged am-Fe(III) >> 3-week aged am-Fe(III) media. The iron uptake rates by C. sociale during 0–1 day in 1 day and 3-day aged am-Fe(III) media were about two-thirds and one-fourths, respectively, lower than that in the direct Fe(III) input medium containing C. sociale into which an acidic Fe(III) stock solution was added directly. The longer aging time of am-Fe(III) in media results in reducing the supply of bioavailable iron in the media by the slower dissolution rate of am-Fe(III) with the longer aging time. These results suggest that the chemical and structural changes of freshly precipitated amorphous ferric hydroxide with short aging time affect their ability, such as iron solubility and dissolution rate to supply bioavailable iron for the phytoplankton growth. The chemical and structural conversion of solid iron phases with time is one of the most important processes in changing the supply of available iron to marine phytoplankton in estuarine and coastal waters and in iron fertilization experiments.

Increased Divalent Metal Transporter 1 Expression Might Be Associated with the Neurotoxicity of l-DOPA

Based on the available data, we speculated that changes in brain iron metabolism induced by L-DOPA might be associated with the neurotoxicity of L-DOPA. To investigate this possibility, the effects of L-DOPA on the expression of iron influx proteins [transferrin receptor (TfR) and divalent metal transporter 1 (DMT1)], iron efflux protein (ferroportin 1), and iron uptake in C6 glioma cells were determined in this study using Northern blot and Western blot analysis and the calcein method. The findings showed that treatment of C6 cells with different concentrations of L-DOPA (0-100 microM) did not affect the expression of mRNA and protein of TfR and DMT1 with iron-responsive element (+IRE) and protein of ferroportin 1. However, a significant increase in the expression of DMT1(-IRE) mRNA and protein was found in cells treated, respectively, with 10 and 30 microM L-DOPA (mRNA) and 1, 5, 10 and 30 microM L-DOPA (protein). The increase in DMT(-IRE) protein induced by L-DOPA treatment was in parallel with the increase in DMT(-IRE) mRNA. The levels of DMT1(-IRE) mRNA and protein peaked in the cells treated with 10 microM L-DOPA and then decreased progressively with increasing concentrations of L-DOPA. Further study demonstrated that treatment of the cells with 10 microM L-DOPA induced a significant increase in ferrous uptake by C6 glioma cells. The findings suggested that the increased DMT1(-IRE) expression might be partly associated with the neurotoxicity of L-DOPA. Clinical relevance of the findings needs to be investigated further.

Antagonistic Roles of ERK1/2 and p38 MAP Kinases in Hemoglobin Synthesis.

Murine erythroleukemia (MEL) cells provide a valuable model to study the molecular events leading to erythroid differentiation. Maturing erythroid cells synthesize large quantities of hemoglobin, a process requiring the coordinated synthesis of heme and globin. Here, we investigated the role of the ERK and p38 mitogen-activated protein kinase (MAPK) signaling pathways in the differentiation of MEL cells. We determined the effect of the MEK1/2 inhibitor U0126 that blocks the ERK1/2 pathway, and the p38 inhibitor SB202190 on the differentiation potential of MEL cells induced by hexamethylene bisacetamide (HMBA). We found that treatment of HMBA induced MEL cells with the ERK1/2 pathway inhibitor U0126 results in higher hemoglobin levels. Using a fluorometric assay, we determined that intracellular heme levels also increased. Immunoblot studies showed an increase in globin protein levels. In contrast, treatment of MEL cells with the p38 inhibitor SB202190 has the opposite effect, leading to decreased amounts of heme and hemoglobin. In addition, inhibition of the p38 pathways results in lower transferrin receptor levels. Our results suggest that the ERK1/2 and p38 pathways play antagonistic roles in HMBA induced erythroid differentiation in MEL cells. This data also provides a novel link between MAPK signaling and the regulation of heme biosynthesis and iron uptake in erythroid cells.

Nm1054: a spontaneous, recessive, hypochromic, microcytic anemia mutation in the mouse

AbstractHypochromic, microcytic anemias are typically the result of inadequate hemoglobin production because of globin defects or iron deficiency. Here, we describe the phenotypic characteristics and pathogenesis of a new recessive, hypochromic, microcytic anemia mouse mutant, nm1054. Although the mutation nm1054 is pleiotropic, also resulting in sparse hair, male infertility, failure to thrive, and hydrocephaly, the anemia is the focus of this study. Hematologic analysis reveals a moderately severe, congenital, hypochromic, microcytic anemia, with an elevated red cell zinc protoporphyrin, consistent with functional erythroid iron deficiency. However, serum and tissue iron analyses show that nm1054 animals are not systemically iron deficient. From hematopoietic stem cell transplantation and iron uptake studies in nm1054 reticulocytes, we provide evidence that the nm1054 anemia is due to an intrinsic hematopoietic defect resulting in inefficient transferrin-dependent iron uptake by erythroid precursors. Linkage studies demonstrate that nm1054 maps to a genetic locus not previously implicated in microcytic anemia or iron phenotypes.

Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells

The reduction of iron is an essential step in the transferrin (Tf) cycle, which is the dominant pathway for iron uptake by red blood cell precursors. A deficiency in iron acquisition by red blood cells leads to hypochromic, microcytic anemia. Using a positional cloning strategy, we identified a gene, six-transmembrane epithelial antigen of the prostate 3 (Steap3), responsible for the iron deficiency anemia in the mouse mutant nm1054. Steap3 is expressed highly in hematopoietic tissues, colocalizes with the Tf cycle endosome and facilitates Tf-bound iron uptake. Steap3 shares homology with F(420)H(2):NADP(+) oxidoreductases found in archaea and bacteria, as well as with the yeast FRE family of metalloreductases. Overexpression of Steap3 stimulates the reduction of iron, and mice lacking Steap3 are deficient in erythroid ferrireductase activity. Taken together, these findings indicate that Steap3 is an endosomal ferrireductase required for efficient Tf-dependent iron uptake in erythroid cells.

Antisense gene delivered by an adenoassociated viral vector inhibits iron uptake in human intestinal cells: Potential application in hemochromatosis

Hereditary hemochromatosis (HH) is a condition in which intestinal iron absorption is greatly elevated. Present treatment is weekly phlebotomy, affecting quality of life and leading to recurrent infections. The iron transporter divalent metal transporter-1 (DMT-1) of enterocytes is responsible for iron uptake from the intestinal lumen; iron is further extruded into the blood by the basolateral transporter ferroportin-1. A therapeutic approach for HH could start with a long-term reduction of iron transport by reduction of DMT-1 levels. We designed an AAV vector coding for a short antisense RNA (AAV-DMT-1-AS) against DMT-1, which reduced iron uptake by 50–60% in human intestinal cells (Caco-2). At low infection levels, DMT-1 mRNA virtually disappeared, suggesting RNAi-like and/or RNase H antisense effects. DMT-1 mRNA levels returned to normal at higher infection levels, indicating that an additional mechanism of mRNA occupation, able to block DMT-1 translation and to avoid feedback regulation by iron responsive elements (IRE), also exists. Cell morphology was normal in all cases and no increases in the interferon-related responses, measured by (a) 2′-5′ A oligo synthetase (b) IFITM1 and (c) ISGF3γ mRNA levels, were observed. Studies presented herein indicate that enterocyte targeting with a gene coding for a short antisense against iron transport blocks enterocyte iron uptake, which may have therapeutic value.

Virulence-associated traits in avian Escherichia coli: Comparison between isolates from colibacillosis-affected and clinically healthy layer flocks

Colibacillosis appears to be of increasing importance in layer flocks. The aim of this study was to determine characteristics of avian pathogenic Escherichia coli associated with the occurrence of colibacillosis outbreaks at flock level. Forty E. coli strains originating from layers from healthy flocks ('control isolates'), consisting of 25 caecal and 15 extra-intestinal isolates, were compared with 40 strains isolated from layers originating from colibacillosis-affected flocks ('outbreak isolates'), consisting of 20 caecal and 20 extra-intestinal isolates. The examined characteristics were adhesins, invasivity in T84 cell culture, serum resistance, iron uptake, colicin production, and toxinogenicity. The following traits were significantly more often detected in the outbreak isolates than in the control isolates: tsh, iss, iucA, iutA, irp2, fyuA, iroC, cvaC, colicin and colicin V production. A comparison of the extra-intestinal outbreak isolates and the caecal control isolates yielded the same results as when the caecal isolates, extra-intestinal isolates and total number of isolates of the outbreak and the control group were compared. When comparing the caecal and extra-intestinal isolates within the control and within the outbreak group, no significant differences were detected. The O78 and O2 groups showed significant differences with other O-types and NT strains for prevalence of most of the same characteristics. The combination of type 1 fimbriae, tsh, serum resistance, iss, traT, iucA, fyuA, iroC and colicin or colicin V production was significantly more often present in extra-intestinal outbreak isolates than in extra-intestinal control isolates. Only the combination of serum resistance, fyuA and colicin production was present in all outbreak isolates, with a significantly lower prevalence in the control isolates. None of the characteristics or combinations examined were exclusive to the outbreak isolates.

Iron uptake and toxicity in Caco-2 cells

The differences between the in vitro effects of iron attributed to valence, chelation, and complexation are known in terms of markers of oxidative stress. Few studies, however, describe the effects of iron on general markers of toxicity used in the testing of cell cultures. The aim of the present study was to determine the toxicity and uptake of different salts and iron complexes in the human intestinal cell line, Caco-2. Cells were incubated with 1.5 mM of different species of iron [FeCl 3/nitrilotriacetic acid (NTA) (1:2), FeCl 3/citric acid (1:2), FeCl 3 and FeSO 4] for 22–24 h. Thereafter, toxicological and uptake experiments were performed. The iron uptake, viability (via MTT assay), and membrane stability (via LDH release) of Caco-2 cells incubated with various iron forms differed significantly from untreated controls which showed no detrimental effects on cells and less iron uptake. The lowest signal for cell viability (MTT assay) was found after the incubation of the cells with FeCl 3/citric acid, being significantly different to treatment with FeCl 3, where the highest MTT signal was detected ( p=0.002). No differences between the tested iron species could be found regarding cell proliferation (via serial cell counting) and viability using the trypan blue exclusion test. The lowest membrane damage (via LDH release) was registered in cells treated with FeCl 3/citric acid (1:2), whereas the highest LDH release could be found in cells incubated with FeCl 3/NTA (1:2). The highest intracellular iron concentration (measured via GFAAS) was detected after the treatment of Caco-2 cells with FeCl 3 and FeCl 3/NTA (1:2). This study substantiates the importance of the choice of complexes, as NTA seemed to enhance the toxicity of iron, while citric acid inhibited iron uptake and toxicity.

Parenteral iron nephrotoxicity: Potential mechanisms and consequences

Parenteral iron administration is a mainstay of anemia management in renal disease patients. However, concerns of potential iron toxicity persist. Thus, this study was conducted to more fully gauge iron toxicologic profiles and potential determinants thereof. Isolated mouse proximal tubule segments (PTS) or cultured proximal tubular [human kidney (HK-2)] cells were exposed to four representative iron preparations [iron sucrose (FeS), iron dextran (FeD), iron gluconate (FeG), or iron oligosaccharide (FeOS)] over a broad dosage range (0, 30 to 1000 microg iron/mL). Cell injury was assessed by lactate deyhdrogenase (LDH) release, adenosine triphosphate (ATP) reductions, cell cytochrome c efflux, and/or electron microscopy. In vivo toxicity (after 2 mg intravenous iron injections) was assessed by plasma/renal/cardiac lipid peroxidation [malondialdehyde (MDA)], renal ferritin (protein)/heme oxygenase-1 (HO-1) (mRNA) expression, electron microscopy, or postiron injection PTS susceptibility to attack. In each test, iron evoked in vitro toxicity, but up to 30x differences in severity (e.g., ATP declines) were observed (FeS > FeG > FeD = FeOS). The in vitro differences paralleled degrees of cell (HK-2) iron uptake. In vivo correlates of iron toxicity included variable increases in renal MDA, ferritin, and HO-1 mRNA levels. Again, these changes appeared to parallel in vivo (glomerular) iron uptake (seen with FeS and FeG, but not with FeD or FeOS). Iron also effected in vivo alterations in proximal tubule cell homeostasis, as reflected by the "downstream" emergence of tubule resistance to in vitro oxidant attack. Parenteral iron formulations have potent, but highly variable, cytotoxic potentials which appear to parallel degrees of cell iron uptake (FeS > FeG >> FeD or FeOS). That in vitro injury can be expressed at clinically relevant iron concentrations, and that in vivo glomerular iron deposition/injury may result, suggest caution is warranted if these agents are to be administered to patients with active renal disease.

Caco-2 cell iron uptake from human milk and infant formula

Knowledge of the nutritional quality of iron in human milk and infant formula is of great interest to nutritionists and food scientists. In this paper we review the reasons for a larger fractional (but not absolute) iron absorption from human milk than from infant formulas. We also present the results of an in vitro digestion/Caco-2 cell model system comparing the cell iron uptake of different infant formulas to that from human milk.