scholarly journals In Vitro Bioaccessibility and Bioavailability of Iron from Mature and Microgreen Fenugreek, Rocket and Broccoli

Nutrients ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 1057
Author(s):  
Kholoud K. Khoja ◽  
Amy Buckley ◽  
Mohamad F. Aslam ◽  
Paul A. Sharp ◽  
Gladys O. Latunde-Dada
Keyword(s):  
Iron Deficiency ◽  
Iron Uptake ◽  
Mineral Content ◽  
Microwave Digestion ◽  
Surrogate Marker ◽  
Risk Groups ◽  
Ammonium Citrate ◽  
Iron Nutrition ◽  
Ferric Ammonium Citrate

Iron deficiency is a global epidemic affecting a third of the world’s population. Current efforts are focused on investigating sustainable ways to improve the bioavailability of iron in plant-based diets. Incorporating microgreens into the diet of at-risk groups in populations could be a useful tool in the management and prevention of iron deficiency. This study analysed and compared the mineral content and bioavailability of iron from microgreen and mature vegetables. The mineral content of rocket, broccoli and fenugreek microgreens and their mature counterparts was determined using microwave digestion and ICP-OES. Iron solubility and bioavailability from the vegetables were determined by a simulated gastrointestinal in vitro digestion and subsequent measurement of ferritin in Caco-2 cells as a surrogate marker of iron uptake. Iron contents of mature fenugreek and rocket were significantly higher than those of the microgreens. Mature fenugreek and broccoli showed significantly (p < 0.001) higher bioaccessibility and low-molecular-weight iron than found in the microgreens. Moreover, iron uptake by Caco-2 cells was significantly higher only from fenugreek microgreens than the mature vegetable. While all vegetables except broccoli enhanced FeSO4 uptake, the response to ferric ammonium citrate (FAC) was inhibitory apart from the mature rocket. Ascorbic acid significantly enhanced iron uptake from mature fenugreek and rocket. Microgreen fenugreek may be bred for a higher content of enhancers of iron availability as a strategy to improve iron nutrition in the populace.

Chemicals That Stimulate Iron Uptake – A Novel Approach to Treatment of Iron Deficiency,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3180-3180
Author(s):  
Zhen Li
Keyword(s):  
Iron Deficiency ◽  
Small Molecules ◽  
Iron Uptake ◽  
Iron Absorption ◽  
K562 Cells ◽  
Divalent Metal ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Caco2 Cells ◽  
Ferric Ammonium

Abstract 3180 Iron (Fe) is an essential nutrient required for all cells, especially for erythrocyte hemoglobin synthesis which requires absorption of 1–2 mg of iron from the gastrointestinal tract. Iron deficiency as a result of inadequate dietary uptake has multiple consequences including anemia and abnormal neurologic development in children and is a global public health concern. Enterocytes in the duodenum, the site of iron absorption, can extract about 10% of dietary Fe. Nonetheless for numerous reasons simple iron supplementation has not solved the worldwide epidemic of iron deficiency. We hypothesized that small molecules which could potentiate iron uptake into cells would allow enterocytes to absorb an increased amount of dietary iron and could be beneficial in limiting iron deficiency. To identify molecules that would accelerate Fe uptake we used a high through-put screening system in conjunction with a reporter system of K562 cells loaded with the divalent metal chelator calcein whose fluorescence is quenched with chelation of Fe2+. Small molecules that stimulated Fe uptake were defined as causing increased calcein fluorescence quenching compared to Fe alone. K562 cells were exposed to 0.1 μM calcein for 10 minutes, thoroughly washed, and 1 × 105 cells plated into each well of multiple 96-well plates. After equilibration of the plates at 37° C, aliquots of the individual components of an in-house chemical library of ∼12,000 compounds dissolved in DMSO were screened in duplicate or triplicate and fluorescence measurements made at 0 and 30 min after addition of 10 μM FeNH4SO4 in a Synergy IV plate reader. 30 chemicals were identified that stimulated iron-induced quenching of calcein fluorescence. The stimulation was verified by dose response curves and by assaying the effect on non-transferrin bound 55Fe uptake. None of the stimulators were cytotoxic for up to at least 3 days. The lead compound, LS081, had an IC50 = 1.22 ± 0.48 μM for 55Fe uptake in K562 cells compared to controls. LS081 was also used to examine the iron uptake in Caco2 cells grown in bicameral chambers, a model system to study intestinal iron absorption. LS081 significantly increased 55Fe uptake into Caco2 cells with a very rapid influx of 55Fe in the first 5 min after Fe was offered to the apical surface followed by a ∼ 4-fold increased uptake over the next 90 min. 55Fe transport across the basolateral surface into the basal chamber also increased ∼ 4 fold. The increased 55Fe transport in caco2 cells is more prominent at lower pH of 5.5 compare to pH 7.5 suggesting LS081 acted on a common divalent metal uptake pathway. Mice treated with LS081 + ferric ammonium citrate via oral gavage for two weeks significantly increased (p < 0.001 by unpaired t-test compared to ferric ammonium citrate alone) the level of ferritin, the iron storage protein, in the liver, demonstrating the absorption of LS081 from intestinal cells. In summary, using high through-put screening technique we identified small molecules that stimulate iron uptake and could be used as a drug for iron deficiency. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Study on Sucrosomial® Iron Endocytosis-Mediated Uptake and Enterocytes Release

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4892-4892
Author(s):  
Elisa Brilli ◽  
Asperti Michela ◽  
Magdalena Gryzik ◽  
Alessandro Lucchesi ◽  
Giovanni Martinelli ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Iron Uptake ◽  
Iron Homeostasis ◽  
Iron Absorption ◽  
Phospholipid Bilayer ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Iron Release ◽  
Iron Storage

Abstract Introduction: iron homeostasis is maintained by regulating the iron levels in plasma which is maintained by four coordinated processes: duodenal iron absorption, macrophage iron recycling, hepatic iron storage and erythropoiesis. Iron in the Fe2+ form is transported across the apical duodenal membrane by DMT1 and subsequently transferred to the blood via the iron exporter, Ferroportin the only know cell membrane iron exporter. Due to the presence of two check points at cellular levels, iron absorption and release are mainly regulated, because of this iron containing oral formulations are poorly absorbed and bioavailable. To overcome cellular barriers and increasing the bioavailability of supplemented iron forms, there is a need for new carriers that work protecting the iron as well as enhancing its intestinal absorption and release into the blood stream. Moreover thus reducing dosage and side effects. Sucrosomial® Iron (SI) represents an innovative oral iron-containing carrier in which ferric pyrophosphate is protected by a phospholipid bilayer membrane plus a sucrester matrix. To date, in vitro studies have shown that SI is mostly absorbed as vesicle-like structure, bypassing the conventional iron absorption pathway. Due to its behaviour at the gastrointestinal tract, SI is well tolerated and highly bioavailable compared to conventional iron salts. To deeply understand involvement of endocytosis in SI absorption and release, in vitro experiments using endocytosis and ferroportin inhibitors were carried out Aim: to study Sucrosomial® Iron uptake and release in different in vitro systems. Materials and Methods: CACO-2 and THP1 cells were used to investigate the role of FPN in Sucorsomial Iron release from cells. For release study, CACO-2 cells were exposed for 18h to quercetin (150mmol/L) in order to downregulate FPN expression. CACO-2 quercetin pre-treated cells were co-cultured with TPH1 cells, and SI or FAC were added. However, prior to measure cell Ferritin content, the incubation medium was discarded and cells were washed to remove quercetin. Iron uptake-release analysis was performed using co-culture transwell system between CACO-2 cells and TPH1. To investigate the cellular fate of cellular iron in quercetin treated CACO-2 and TPH1 cells we measured cell ferritin content. To inhibit endocytosis absorption pathway, CACO-2, THP1 and HepG2 cells were pre-treated with PitStop2 and Dyngo 4a inhibitors and then treated with SI, or Ferrous Sulfate (FS) or ferric ammonium citrate (FAC). Cellular Ferritin content was measured. Results: in order to understand the effect of quercetin on iron storage, we used CACO2 and TPH1 cells pre-treated with quercetin and then treated with SI, FAC or nothing (control). Quercetin-SI treated CACO-2 cells showed no differences in Ferritin expression compared to control cells (3,94 ngFTL/mg proteins Vs 4,56 ngFTL/mg proteins) while in quercetin-FAC treated cells ferritin expression was decreased compare to control cells (16,3 ngFTL/mg proteins Vs 27,55 ngFTL/mg proteins). In a similar manner, quercetin-SI treated TPH1 cells didn't show increase in Ferritin expression compared to control cells (20 ngFTL/mg proteins Vs 15,15 ngFTL/mg proteins), only in quercetin-FAC treated cells we observed a Ferritin expression increase compared to control untreated cells (16 ngFTL/mg proteins Vs 24 ngFTL/mg proteins). Results from experiments using endocytosis inhibitors showed that SI absorption in CACO-2 cells is inhibited using Dyngo4a (from 4ngFTL/mg proteins to 0,36 ngFTL/ mg proteisn) while PitStop3 seems to reduce SI absorption in THP1 (from 396 ngFL/mg protein to 199,91 ngFTL/mg proteins) and HepG2 cells (from 26,86 ngFL/mg proteins to 3,93 ngFTL/mg proteins), since ferritin expression significantly decrease only in SI treated cells. Conclusions: endocytosis pathway seems to be involved in SI cellular uptake but this process is regulated in different manner probably due to different cell types. Release experiments showed that cells treated with quercetin could reduce for a negative feedback DMT1 expression as well, affecting iron uptake from cells treated with FAC but not with SI and consequently, if SI is able to bypass commonly iron uptake mechanism, FPN inhibition did not show iron release perturbation from cells treated with SI. Disclosures Brilli: Pharmanutra s.p.a.: Consultancy. Martinelli:Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Roche: Consultancy; Abbvie: Consultancy; Novartis: Speakers Bureau; Amgen: Consultancy; Ariad/Incyte: Consultancy; Jazz Pharmaceuticals: Consultancy. Tarantino:Pharmanutra s.p.a.: Employment.

scholarly journals Ankaferd Influences mRNA Expression of Iron-Regulated Genes During Iron-Deficiency Anemia

2017 ◽  
Vol 24 (6) ◽  
pp. 960-964 ◽  
Author(s):  
Afife Gulec ◽  
Sukru Gulec
Keyword(s):  
Iron Deficiency ◽  
Iron Deficiency Anemia ◽  
Messenger Rna ◽  
In Vitro Models ◽  
Ammonium Citrate ◽  
Deficiency Anemia ◽  
Ferric Ammonium Citrate ◽  
Marker Genes ◽  
Divalent Metal Transporter

Ankaferd Blood Stopper (ABS) comprises a mixture of plants and stops bleeding via forming a protein network by erythroid aggregation. Bleeding causes reduction of iron levels in body. It has been indicated that ABS contains significant amount of iron. Thus, we investigated the biological activity of ABS-derived iron on iron-regulated genes during iron-deficiency anemia (IDA). IDA We selected Caco-2 and HepG2 cell lines as in vitro models of human intestine and liver, respectively. Iron deficiency anemia was induced by deferoxamine. The cells were treated with ferric ammonium citrate (FAC) and ABS. Messenger RNA levels of iron-regulated genes were analyzed by quantitative reverse transcription polymerase chain reaction to elucidate whether iron in ABS behaved similar to inorganic iron (FAC) during IDA. The results showed that ABS-derived iron influenced transcriptions of iron-regulated marker genes, including divalent metal transporter ( Dmt1), transferrin receptor ( TfR), ankyrin repeat domain 37 ( Ankrd37), and hepcidin ( Hamp) in IDA-induced Caco-2 and HepG2 cells. Our results suggest that when ABS is used to stop tissue bleeding, it might have an ability to reduce levels of IDA.

scholarly journals NCOA4 Mediates Ferritin Responses to Iron, Erythrophagocytosis, and Hepcidin in Macrophages (P24-056-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Cole Guggisberg ◽  
Moon-Suhn Ryu
Keyword(s):  
Iron Homeostasis ◽  
Primary Source ◽  
Viable Cell ◽  
Iron Chelator ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Anemia Of Chronic Disease ◽  
Cell Counts ◽  
Subsequent Decrease

Abstract Objectives Iron recycled from erythrophagocytosis by macrophages serves as a primary source of systemic iron. NCOA4 mediates ferritin turnover via ferritinophagy. Yet, whether NCOA4 is important in macrophages or erythrophagocytosis-mediated iron recycling remains unclear, and thus was assessed in vitro. Methods J774 cells were employed as an in vitro model of macrophages. Iron studies involved treatments of ferric ammonium citrate (FAC) or an iron chelator, deferoxamine (Dfo). To recapitulate systemic iron recycling and overload, cells were treated with opsonized erythrocytes and minihepcidin, respectively. NCOA4 knock-down was achieved by siRNA transfection. Iron gene responses were measured by qPCR and western analyses, and viable cell counts were colorimetrically determined by CCK8 assays as functional outcomes. Results NCOA4 protein abundance was inversely related to iron availability and ferritin in macrophages. Loss of NCOA4 resulted in impaired ferritin turnover, and led to a reduction in viable cells when combined with iron deficiency. By erythrophagocytosis, a peak in ferritin abundance was observed at 12 h with a subsequent decrease at 24 h. This loss in ferritin was NCOA4-dependent. Minihepcidin caused accumulation of ferritin, along with a repression of NCOA4 in both control and erythrocyte-laden macrophages. Hepcidin activity had no effect on ferritin when NCOA4 was depleted. Conclusions NCOA4 mediates the release of ferritin iron during cellular iron restriction and iron recycling by macrophages. Moreover, our studies suggest that macrophage NCOA4 is integral to systemic iron homeostasis by responding to the iron regulatory hormone, hepcidin. Thus, NCOA4 and ferritinophagy may potentially serve as therapeutic targets for treatments of iron disorders and anemia of chronic disease. Funding Sources Supported by the NIFA, USDA, Hatch project under MIN-18–118 and intramural support to M-S.R.

scholarly journals Iron Causes Lipid Oxidation and Inhibits Proteasome Function in Multiple Myeloma Cells: A Proof of Concept for Novel Combination Therapies

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 970 ◽  
Author(s):  
Jessica Bordini ◽  
Federica Morisi ◽  
Fulvia Cerruti ◽  
Paolo Cascio ◽  
Clara Camaschella ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Iron Toxicity ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Ferric Carboxymaltose ◽  
High Dose ◽  
Iron Loading

Adaptation to import iron for proliferation makes cancer cells potentially sensitive to iron toxicity. Iron loading impairs multiple myeloma (MM) cell proliferation and increases the efficacy of the proteasome inhibitor bortezomib. Here, we defined the mechanisms of iron toxicity in MM.1S, U266, H929, and OPM-2 MM cell lines, and validated this strategy in preclinical studies using Vk*MYC mice as MM model. High-dose ferric ammonium citrate triggered cell death in all cell lines tested, increasing malondialdehyde levels, the by-product of lipid peroxidation and index of ferroptosis. In addition, iron exposure caused dose-dependent accumulation of polyubiquitinated proteins in highly iron-sensitive MM.1S and H929 cells, suggesting that proteasome workload contributes to iron sensitivity. Accordingly, high iron concentrations inhibited the proteasomal chymotrypsin-like activity of 26S particles and of MM cellular extracts in vitro. In all MM cells, bortezomib-iron combination induced persistent lipid damage, exacerbated bortezomib-induced polyubiquitinated proteins accumulation, and triggered cell death more efficiently than individual treatments. In Vk*MYC mice, addition of iron dextran or ferric carboxymaltose to the bortezomib-melphalan-prednisone (VMP) regimen increased the therapeutic response and prolonged remission without causing evident toxicity. We conclude that iron loading interferes both with redox and protein homeostasis, a property that can be exploited to design novel combination strategies including iron supplementation, to increase the efficacy of current MM therapies.

scholarly journals Persistence of contamination of hens' egg albumen in vitro with Salmonella serotypes

1992 ◽  
Vol 108 (3) ◽  
pp. 389-396 ◽  
Author(s):  
J. L. Lock ◽  
R. G. Board
Keyword(s):  
Cell Membrane ◽  
Glucose Concentration ◽  
Salmonella Enteritidis ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Egg Albumen ◽  
Ferric Ammonium ◽  
Salmonella Serotypes

SUMMARYA study was made of the persistence of different Salmonella serotypes in hens' egg albumen in vitro at 4, 20 and 30 °C. The majority of serotypes remained viable but did not increase in numbers at 20 and 30 °C for 42 days. At 4 °C many of the serotypes died out.The addition of ferric ammonium citrate on the 42nd day of incubation induced multiplication of organisms incubated at 20 and 30 °C, but not at 4 °C. The pH and glucose concentration of the albumen diminished only when heavy growth occurred.Salmonella enteritidis remained viable on the air cell membrane in vitro for 17 days at 4, 20 and 30 °C. Thirty percent of the organisms also remained motile in albumen for 42 days at 25 °C and up to 5% of the cells remained motile for up to 20 days at 4 °C.

Sucrosomial Iron®: A New Highly Bioavaible Oral Iron Supplement

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4561-4561 ◽  
Author(s):  
Germano Tarantino ◽  
Elisa Brilli ◽  
Ylenia Zambito ◽  
Giulio Giordano ◽  
Francesco Equitani
Keyword(s):  
Iron Deficiency ◽  
Iron Uptake ◽  
Iron Bioavailability ◽  
Oral Iron ◽  
Deficiency Anemia ◽  
Sucrose Esters ◽  
Absorption Enhancers ◽  
Ferric Pyrophosphate ◽  
Ferritin Synthesis

Abstract Introduction: Iron deficiency is one of the most widespread nutritional deficiencies. Globally two billion people are suffering from iron- deficiency anemia (Hermida et al., 2010). Oral therapy for iron deficiency is mainly based on immediate release formulations of ferrous iron. However, modified formulations have been marketed to reduce gastrointestinal side effects and to prevent iron instability in the gastrointestinal tract. Overcoming biological barriers, including the gastrointestinal epithelial barriers, is a great challenge for pharmaceutical research and thus there is a need for new absorption enhancers with more favorable profile. Sucrose esters are widely used in the food industry, and there are reports on their potential use in pharmaceutical formulations as excipients (Szuts A et al., 2008). In vitro methods using cell cultures have been proposed to assess iron bioavailability as an alternative to in vivo methods. Caco-2 cells have shown numerous morphological and biochemical characteristics of enterocytes and have been successfully used to study iron absorption (Garcia et al., 1996; Jovani et al., 2001). Caco-2 monolayers formed a good barrier as reflected by high transepithelial resistance and positive immunostaining for junctional proteins. Sucrose esters in nontoxic concentrations significantly reduced resistance and impedance, and increased permeability of some components in Caco-2 monolayers. Recent data indicate that sucrose esters can enhance drug permeability through both the transcellular and paracellular routes (Kiss et al., 2014). Aim: The strong correlation between the published human absorption data and the iron uptake by Caco-2 cells makes them an ideal in vitro model to study iron bioavailability (Au and Reddy, 2000). For this, in the present study, we compared the bioavailability of innovative Oral Iron formulation based on Sucrosomial Iron¨ (Sideral¨) with three different Iron formulations (Figure 1). Materials and Methods: Sucrosomial Iron, preparation of ferric pyrophosphate convered by a phospholipids plus sucrose esters of fatty acids matrix; Lipofer¨, a water-dispersible micronised iron; Sunactive¨ ferric pyrophosphate, lecithin and emulsifiers. Results: The data showed that Sucrosomial Iron¨ (Sideral¨), is significantly more bioavaible than microencapsulated Ferric pyrophosphate ingredients, Lipofer¨ and Sunactive¨ and Ferrous Sulfate in Caco-2 cell model (Figure 1). Bibliography Au, A. P., Reddy, M. B. (2000). Caco-2 cells can be used to assess human iron bioavailability from a semipurified meal. J Nutr 130:1329-1334. Garcia et al. (1996). The Caco-2 cell culture system can be used as a model to study food iron availability. J Nutr 126:251-258. Hermida et al., Preparation and characterization of iron-containing liposomes: their effect on soluble iron uptake by Caco-2 cells Journal of Liposome Research, 2010, 1-10, Jovani et al. (2001) Calcium, iron, and zinc uptake from digests of infant formulas by Caco-2 cells. J Agric Food Chem 49:3480-3485. Kiss et al., (2014) Sucrose esters increase drug penetration, but do not inhibit p-glycoprotein in caco-2 intestinal epithelial cells J Pharm Sci. Oct;103(10):3107-19. Szuts A et al. (2008) Study of the effects of drugs on the structures of sucrose esters and the effects of solid-state interactions on drug release J Pharm Biomed Anal. 48: Figure 1. the graph shows the Ferritin levels of Caco-2 cells after iron formulations treatment. Sucrosomial Iron treated cells display significant increase of Ferritin synthesis compared to Lipofer and SunActive treated cells. Figure 1. the graph shows the Ferritin levels of Caco-2 cells after iron formulations treatment. Sucrosomial Iron treated cells display significant increase of Ferritin synthesis compared to Lipofer and SunActive treated cells. Disclosures Tarantino: Pharmanutra s.p.a.: Employment. Brilli:Pharmanutra s.p.a.: Employment.

scholarly journals Ferric citrate decreases ruminal hydrogen sulphide concentrations in feedlot cattle fed diets high in sulphate

2013 ◽  
Vol 111 (2) ◽  
pp. 261-269 ◽  
Author(s):  
Mary E. Drewnoski ◽  
Perry Doane ◽  
Stephanie L. Hansen
Keyword(s):  
Hydrogen Sulphide ◽  
Rumen Fluid ◽  
Control Diet ◽  
Gas Production ◽  
Feedlot Cattle ◽  
Ferric Citrate ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Ferric Ammonium

Dissimilatory reduction of sulphate by sulphate-reducing bacteria in the rumen produces sulphide, which can lead to a build-up of the toxic gas hydrogen sulphide (H2S) in the rumen when increased concentrations of sulphate are consumed by ruminants. We hypothesised that adding ferric Fe would competitively inhibit ruminal sulphate reduction. The effects of five concentrations and two sources (ferric citrate or ferric ammonium citrate) of ferric Fe were examinedin vitro(n6 per treatment). Rumen fluid was collected from a steer that was adapted to a high-concentrate, high-sulphate diet (0·51 % S). The addition of either source of ferric Fe decreased (P< 0·01) H2S concentrations without affecting gas production (P= 0·38), fluid pH (P= 0·80) orin vitroDM digestibility (P= 0·38) after a 24 h incubation. Anin vivoexperiment was conducted using eight ruminally fistulated steers (543 (sem12) kg) in a replicated Latin square with four periods and four treatments. The treatments included a high-concentrate, high-sulphate control diet (0·46 % S) or the control diet plus ferric ammonium citrate at concentrations of 200, 300 or 400 mg Fe/kg diet DM. The inclusion of ferric Fe did not affect DM intake (P= 0·21). There was a linear (P< 0·01) decrease in the concentration of ruminal H2S as the addition of ferric Fe concentrations increased. Ferric citrate appears to be an effective way to decrease ruminal H2S concentrations, which could allow producers to safely increase the inclusion of ethanol co-products.

scholarly journals NCOA4-Mediated Ferritinophagy Is Essential for HT22 Neuronal Cell Survival During Iron Deficiency (OR15-07-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Emily Bengson ◽  
Moon-Suhn Ryu
Keyword(s):  
Cell Death ◽  
Iron Deficiency ◽  
Iron Overload ◽  
Cell Survival ◽  
Neuronal Cell ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Mrna Levels ◽  
Ht22 Cells ◽  
Cellular Iron

Abstract Objectives Iron is essential for proper cell function and development. However, mishandled iron may lead to ferroptotic cell death. NCOA4 manages the cellular labile iron pool by controlling the release of ferritin iron via ferritinophagy. The present studies examined the capacity of hippocampal cells in handling iron fluctuation, with particular focus on NCOA4 and ferritin. Methods HT22 mouse hippocampal cells were treated with ferric ammonium citrate (FAC) and deferoxamine (Dfo) to produce cellular iron overload and deprivation, respectively. Ferroptosis was determined by measures of ferrostatin-1 effects and Ptgs2 mRNA. For ferritinophagy studies, Ncoa4 was silenced by siRNA transfections. Functional impacts of impaired ferritinophagy were assessed via CCK-8 cell viability assays and western and qPCR analyses of iron-related genes. Results HT22 cells were highly susceptible to cellular iron overload. FAC-treated cells featured acute morphological changes, decreased viability, and elevated Ptgs2 mRNA abundance. Iron effects were prevented by ferrostatin-1, indicating ferroptosis by cellular iron overload. Dfo alone had minimal impact on cell morphology and viability. NCOA4 protein, but not mRNA, levels were acutely upregulated by Dfo treatments. Ferritin turnover by iron deficiency was impaired in NCOA4-depleted cells, presumably due to impaired ferritinophagy. Moreover, HT22 cells became sensitive to iron deficiency by loss of NCOA4. Conclusions Our studies demonstrate iron can induce ferroptosis in neuronal cells. We also identify NCOA4-mediated ferritinophagy as an integral process for neuronal cell survival during iron deficiency. Further investigation using multi-omics approaches are in progress to determine the mechanisms by which NCOA4 depletion leads to cell death when extracellular iron supply is limited. Funding Sources Supported by the NIFA, USDA, Hatch project under MIN-18–118 and intramural support to M-S.R.

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