ferric-ammonium-citrate and Iron-Overload

Oligodendrocytes are the most iron-rich cells in the brain. Studies have shown that oligodendrocytes are very sensitive to oxidative stress, and iron overload is more likely to cause damage to oligodendrocytes. The purpose of this experiment was to investigate the damaging effect and mechanism of ferric ammonium citrate (FAC) on MO3.13 oligodendrocytes. In FAC treatment group, the intracellular iron concentration and intracellular reactive oxygen species were increased. There were no obvious changes in nucleus and chromatin, but increased mitochondrial membrane density, decreased mitochondrial cristae and mitochondrial length were observed. Glutathione peroxidase 4 (GPX4) expression was decreased, but the ratio of Bcl-2/Bax protein levels and cleaved caspase-3 expression did not change. Moreover, the iron chelator deferoxamine (DFO) and the ferroptosis inhibitor ferrostatin-1(Fer-1) could inhibit the upregulation of GPX4, which indicating that DFO and Fer-1 could inhibit ferroptosis in MO3.13 oligodendrocytes induced by iron overload. Furthermore, the phosphorylation level of p53 was not changed, while the ratio of protein expressions of p-Erk1/2/Erk1/2 were markedly increased. Taken together, our data suggest that iron overload induces ferroptosis but not apoptosis in oligodendrocytes. The mechanism may be related to mitogen-activated protein kinase pathway activation rather than p53 pathway activation.

Topics: Apoptosis; Ferroptosis; Humans; Iron; Iron Overload; Reactive Oxygen Species

Iron homeostasis plays a positive role in articular cartilage health. Excessive iron or iron overload can induce oxidative stress damage in chondrocytes and ferroptosis cell death, advancing knee osteoarthritis (KOA). However, up to date, few effective agents treat iron overload-induced KOA (IOKOA). Chinese herbal medicine (CHM) provides abundant resources for drug selection to manage bone metabolic conditions, including osteoporosis. Biochanin A (BCA) is a novel bioactive multifunctional natural compound isolated from Huangqi, which has protective effects on bone loss. Nevertheless, the function and mechanism of BCA in treating IOKOA are still elusive.. This study seeks to uncover the potential therapeutic targets and mechanisms of BCA in the management of KOA with iron accumulation.. Iron dextrin (500 mg/kg) was intraperitoneally injected into mice to establish the iron overloaded mice model. OA was induced through surgery, and the progression was evaluated eight weeks following surgery. OA severity was evaluated with micro-CT and Safranin-O/Fast green staining in vivo. Iron deposition in the knee joint and synovium was assessed using Perl's Prussian blue staining. Ferric ammonium citrate (FAC) was then administered to primary chondrocytes to evaluate iron regulators mediated iron homeostasis. Toluidine blue staining was utilized to identify chondrocytes in vitro. The vitality of the cells was assessed using the CCK-8 test. The apoptosis rate of cells was measured using Annexin V-FITC/PI assay. The intracellular iron level was detected utilizing the calcein-AM test. Reactive oxygen species (ROS), lipid-ROS, and mitochondrial membrane potentiality were reflected via fluorescence density. Utilizing RT-qPCR and western blotting, the expression level was determined.. Micro-CT and histological staining of knee joints showed greater cartilage degradation and higher iron buildup detected in iron-overloaded mice. BCA can reduce iron deposition and the severity of KOA. Toluidine blue staining and the CCK-8 assay indicated that BCA could rescue chondrocytes killed by iron. Cell apoptosis rates were increased due to iron overload but improved by BCA. Further, the intracellular content of iron, ROS, and lipid-ROS was increased with ferric ammonium citrate (FAC) treatment but restored after treatment with different concentrations of BCA. JC-1 staining revealed that BCA could reduce mitochondrial damage induced by iron overload.. Iron overload was shown to promote chondrocyte ferroptosis in vivo and in vitro. Moreover, iron overload suppressed the expression of collagen II and induced MMP expression by catalyzing ROS generation with mitochondrial dysfunction. Our results showed that BCA could directly reduce intracellular iron concentration by inhibiting TfR1 and promoting FPN but also target the Nrf2/system xc-/GPX4 signaling pathway to scavenge free radicals and prevent lipid peroxidation. The results of this research indicate that BCA regulates iron homeostasis during the progression of osteoarthritis, which can open a new field of treatment for KOA.

Topics: Animals; Chondrocytes; Iron; Iron Overload; Lipids; Mice; Osteoarthritis, Knee; Reactive Oxygen Species; Tolonium Chloride

Topics: Adenomyosis; Adult; Autophagy; Beclin-1; Cell Proliferation; Cells, Cultured; Endometriosis; Endometrium; Female; Ferric Compounds; Humans; Iron Overload; Middle Aged; Oxidative Stress; Poly (ADP-Ribose) Polymerase-1; Quaternary Ammonium Compounds; Signal Transduction; Sirtuin 1; Stromal Cells; Young Adult

Hepatic iron overload is a universal phenomenon in patients with myelodysplastic syndromes (MDS) who undergo bone marrow transplantation and may experience the toxicity of peri- and post-bone marrow transplantation. To clarify the mechanisms of iron overload-triggered liver injury, we determined the effects of iron overload on changes in protein phosphorylation in human hepatocyte cell line HH4 in vitro by using a phosphoproteomics approach. The hepatocytes were exposed to high concentrations of ferric ammonium citrate (FAC) to build up an iron overload model in vitro. Changes in protein phosphorylation initiated by iron overloading were studied by 2D-LC/MS. We identified 335 differentially expressed phosphorylated proteins under the condition of excess hepatocyte iron, 11% of which were related to cell cycle progression. The results of phosphoproteomics showed that iron overload induced 10.9 times increase in Thr 14/Tyr 15-phosphorylated Cdk1 in HH4 cells. Flow cytometry analysis revealed that FAC-treated HH4 cells showed significant G2/M phase arrest. Our subsequent RT-PCR and Western blot experiments indicated that FAC-induced G2/M phase arrest was related to the activation of p53-p21-Cdk1, p53-14-3-3 sigma-Cdk1, and 14-3-3 gamma pathway. Our findings demonstrate the first evidence that iron overload causes G2/M arrest in HH4 hepatocytes.

Topics: Apoptosis; Cell Division; Cell Line, Tumor; Ferric Compounds; G2 Phase Cell Cycle Checkpoints; Hepatocytes; Humans; Iron; Iron Overload; Liver; Proteomics; Quaternary Ammonium Compounds; Tumor Suppressor Protein p53

Iron is a key element for systemic oxygen delivery and cellular energy metabolism. Thus regulation of systemic and local iron metabolism is key for maintaining energy homeostasis. Significant changes in iron levels due to malnutrition or hemorrhage, have been associated with several diseases such as hemochromatosis, liver cirrhosis and COPD. Macrophages are key cells in regulating iron levels in tissues as they sequester excess iron. How iron overload affects macrophage differentiation and function remains a subject of debate. Here we used an

Topics: Humans; Iron; Iron Overload; Macrophages; Monocytes; Oxidative Stress; Pulmonary Disease, Chronic Obstructive; Reactive Oxygen Species

Iron overload can induce oxidative stress, thereby inducing cell peroxidation. Arachidonic acid (ARA) is widely expressed in mammalian cells and esterified to membrane phospholipids. To explore the effect of iron overload on the metabolism of membrane phospholipids MES23.5 cells were treated with various concentrations of ferric ammonium citrate (FAC) to induce oxidative stress. Using UHPLC (I-Class LC, Waters) coupled to a QTRAP (AB Sciex 5500) technology, the contents of 13 substances of ARA and its metabolites were detected. When the cells were given two different concentrations of FAC, we found that both high and low concentrations decrease the expression of ARA (p=0.002, p=0.02) compared with the control group. ARA has three metabolic pathways: the COX pathway, LOX pathway and CYP450 pathway. Compared with the control group, the LTB4 content in the LOX pathway was decreased (p=0.10) after treatment with lowconcentration FAC, while the LTB4 content was increased in the high-concentration treatment group (p=0.06). However, the content of 12S-HETE (p=0.23, p=0.05) in the LOX metabolic pathway decreased with increase of FAC concentration. Similarly, the content of 15S-HETE also decreased with increase of FAC concentration (p=0.17, p=0.02). The other downstream metabolites of ARA, 9S-HODE (p=0.54, p=0.18) and 13S-HODE (p=0.81, p=0.13) were not significantly changed. The contents of thromboxane B2 (TXB2), leukotriene D4 (LTD4), prostaglandin E2 (PGE2), 8-iso-prostaglandin F2α (8-iso-PGF2α), prostaglandin F2α (PGF2α), 6-keto-PGF1α, and prostaglandin D2 (PGD2) were too low to be detected in MES23.5 cells. The above results indicate that oxidative stress caused by iron overload reduces the LOX metabolic pathway of ARA.

Topics: Animals; Arachidonic Acid; Dinoprost; Hydroxyeicosatetraenoic Acids; Iron Overload; Leukotriene B4; Mammals; Oxidative Stress; Phospholipids

Osteoarthritis (OA) is a common joint disease that is characterized by cartilage degradation. Iron deposition in the joints is common during the pathogenic progression of OA and recent studies have indicated that iron overload is an important contributor to OA progression. Calcium chelators have been reported to inhibit iron influx via modulating transferrin receptor protein 1 internalization, and they have been identified as a potential approach to the treatment of iron overload‑induced diseases. The aim of the present study was to investigate the effect of calcium chelators on the progression of iron overload‑induced OA. Primary chondrocytes were treated with various concentrations of ferric ammonium citrate (FAC) to mimic iron overload

Topics: Animals; Apoptosis; Calcium Chelating Agents; Cartilage Diseases; Cartilage, Articular; Cell Survival; Cells, Cultured; Chondrocytes; Egtazic Acid; Ferric Compounds; Iron Overload; Male; Mice; Mice, Inbred C57BL; Mitochondria; Mitochondrial Diseases; Osteoarthritis; Protective Agents; Quaternary Ammonium Compounds; Reactive Oxygen Species

In plasma, iron is normally bound to transferrin, the principal protein in blood responsible for binding and transporting iron throughout the body. However, in conditions of iron overload when the iron-binding capacity of transferrin is exceeded, non-transferrin-bound iron (NTBI) appears in plasma. NTBI is taken up by hepatocytes and other parenchymal cells via NTBI transporters and can cause cellular damage by promoting the generation of reactive oxygen species. However, how NTBI affects endothelial cells, the most proximal cell type exposed to circulating NTBI, has not been explored. We modeled in vitro the effects of systemic iron overload on endothelial cells by treating primary human umbilical vein endothelial cells (HUVECs) with NTBI (ferric ammonium citrate [FAC]). We showed by RNA-Seq that iron loading alters lipid homeostasis in HUVECs by inducing sterol regulatory element-binding protein 2-mediated cholesterol biosynthesis. We also determined that FAC increased the susceptibility of HUVECs to apoptosis induced by tumor necrosis factor-α (TNFα). Moreover, we showed that cholesterol biosynthesis contributes to iron-potentiated apoptosis. Treating HUVECs with a cholesterol chelator hydroxypropyl-β-cyclodextrin demonstrated that depletion of cholesterol was sufficient to rescue HUVECs from TNFα-induced apoptosis, even in the presence of FAC. Finally, we showed that FAC or cholesterol treatment modulated the TNFα pathway by inducing novel proteolytic processing of TNFR1 to a short isoform that localizes to lipid rafts. Our study raises the possibility that iron-mediated toxicity in human iron overload disorders is at least in part dependent on alterations in cholesterol metabolism in endothelial cells, increasing their susceptibility to apoptosis.

Topics: Apoptosis; Cholesterol; Ferric Compounds; Human Umbilical Vein Endothelial Cells; Humans; Iron; Iron Overload; Quaternary Ammonium Compounds; Tumor Necrosis Factor-alpha

As a novel discovered regulated cell death pattern, ferroptosis has been associated with the development of Parkinson's disease (PD) and has attracted widespread attention. Nevertheless, the relationship between ferroptosis and PD pathogenesis is still unclear. This study aims to investigate the effect of iron overload on dopaminergic (DA) neurons and its correlation with ferroptosis. Here we use nerve growth factor (NGF) induced PC12 cells which are derived from pheochromocytoma of the rat adrenal to establish a classical PD in vitro model. We found significantly decreased cell viability in NGF-PC12 cell under ammonium ferric citrate (FAC) administration. Moreover, excessive intracellular iron ions induced the increase of (reactive oxygen species) ROS release as well as the decrease of mitochondrial membrane potential in PC12-NGF cells. In addition, we also found that overloaded iron can activate cell apoptosis and ferroptosis pathways, which led to cell death. Furthermore, MPP-induced PD cells were characterized by mitochondrial shrinkage, decreased expression of glutathione peroxidase 4 (Gpx4) and ferritin heavy chain (FTH1), and increased divalent metal transporter (DMT1) and transferrin receptor 1 (TfR1) expression level. In contrast, Lip-1 and DFO increased the expression level of GPX4 and FTH1 compared to MPP-induced PD cell. In conclusion, we indicated that overloaded intracellular iron contributes to neurons death via apoptosis and ferroptosis pathways, while DFO, an iron chelator, can inhibit ferroptosis in order to protect the neurons in vitro.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Survival; Deferoxamine; Dopaminergic Neurons; Ferric Compounds; Ferroptosis; Humans; Iron Chelating Agents; Iron Overload; Nerve Growth Factor; Neuroprotective Agents; Parkinson Disease, Secondary; Quaternary Ammonium Compounds; Quinoxalines; Rats; Reactive Oxygen Species; Spiro Compounds

Patients with radiotherapy are at significant risks of bone loss and fracture. On the other hand, osteoporosis often occurs in disorders characterized by iron overload. Either ionizing radiation (IR) or iron overload alone has detrimental effects on bone metabolism, but their combined effects are not well defined. In this study, we evaluated the effects of IR on bone loss in an iron-overload mouse model induced by intraperitoneal injection of ferric ammonium citrate (FAC). In the present study, we found that IR additively aggravated iron overload induced by FAC injections. Iron overload stimulated hepcidin synthesis, while IR had an inhibitory effect and even inhibited the stimulatory effects of iron overload. Micro-CT analysis demonstrated that the loss of bone mineral density and bone volume, and the deterioration of bone microarchitecture were greatest in combined treatment group. Iron altered the responses of bone cells to IR. Iron enhanced the responses of osteoclasts to IR with elevated osteoclast differentiation, but did not affect osteoblast differentiation. Our study indicates that IR and iron in combination lead to a more severe impact on the bone homeostasis when compared with their respective effects. IR aggravated iron overload induced bone loss by heightened bone resorption relative to formation. The addictive effects may be associated with the exacerbated iron accumulation and osteoclast differentiation.

Topics: Animals; Bone Density; Disease Models, Animal; Ferric Compounds; Injections, Intraperitoneal; Iron Overload; Male; Mice; Mice, Inbred C57BL; Osteoclasts; Osteogenesis; Quaternary Ammonium Compounds; Radiation, Ionizing

Iron overload affects the cell cycle of various cell types, but the effect of iron overload on human pluripotent stem cells has not yet been reported. Here, we show that the proliferation capacities of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were significantly inhibited by ferric ammonium citrate (FAC) in a concentration-dependent manner. In addition, deferoxamine protected hESCs/hiPSCs against FAC-induced cell-cycle arrest. However, iron overload did not affect pluripotency in hESCs/hiPSCs. Further, treatment of hiPSCs with FAC resulted in excess reactive oxygen species production and DNA damage. Collectively, our findings provide new insights into the role of iron homeostasis in the maintenance of self-renewal in human pluripotent stem cells.

Topics: Apoptosis; Cell Cycle; Cell Proliferation; Deferoxamine; DNA Damage; Ferric Compounds; Humans; Induced Pluripotent Stem Cells; Iron; Iron Overload; Mesenchymal Stem Cells; Oxidative Stress; Pluripotent Stem Cells; Quaternary Ammonium Compounds; Reactive Oxygen Species; Signal Transduction

Iron overload can result in a disorder in glucose metabolism. However, the underlining mechanism through which iron overload induces beta cell death remains unknown.. According to the concentration of ferric ammonium citrate (FAC) and N-acetylcysteine, INS-1 cells were randomly divided into four groups: normal control (FAC 0 μM) group, FAC 80 μM group, FAC 160 μM group, FAC 160μM + NAC group. Cell proliferation was assessed by Cell Counting Kit-8. Reactive oxygen species (ROS) level was further evaluated using flow cytometer with a fluorescent probe. The mitochondrial membrane potential was detected by JC-1 kit, and transmission electron microscopy was used to observe the mitochondrial changes. The related protein expressions were detected by western bolt to evaluate mitophagy status.. It was shown that FAC treatment decreased INS-1 cell viability in vitro, resulted in a decline in mitochondrial membrane potential, increased oxidative stress level and suppressed mitophagy. Furthermore, these effects could be alleviated by the ROS scavenger.. We proved that increased iron overload primarily increased oxidative stress and further suppressed mitophagy via PTEN-induced putative kinase 1/Parkin pathway, resulting in cytotoxicity in INS-1 cells.

Topics: Acetylcysteine; Animals; Cell Proliferation; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Ferric Compounds; Insulin-Secreting Cells; Iron Overload; Membrane Potential, Mitochondrial; Mitochondria; Oxidative Stress; Protein Kinases; Quaternary Ammonium Compounds; Rats; Reactive Oxygen Species; Ubiquitin-Protein Ligases

Iron-mediated oxidative stress has been recognized as one of the leading causes of chronic kidney injury. The effect of L-type calcium channel (LTCC) blocker on iron overload has been shown in cardiomyocytes, liver cells, and nerve cells. So far, few studies have examined whether blockers improve kidney iron-mediated oxidative stress. Yet, the precise mechanism through which blockers regulate kidney iron transport still remains unclear. In the present work, treatment with nifedipine or verapamil decreased oxidative stress and reduced the cell apoptosis-induced by ferric ammonium citrate (P < 0.05), decreased cellular iron contents, and prevented the rising of iron level-induced by ferric ammonium citrate (P > 0.05) in HK-2 and HEK293 cells. Besides, nifedipine and verapamil treatments increased the expression of divalent metal transporter 1, divalent metal transporter ZIP14, and ferroportin1 in HK-2 cells and increased ferroportin1 expression in HEK293 cells. In summary, LTCC blockers alleviate iron overload-induced oxidative stress in renal epithelial cells by blocking the iron uptake and enhancing cellular iron transport and/or iron export, thus synergistically reducing the cellular iron accumulation. Consequently, LTCC blockers may be used as a novel treatment for the prevention of primary or secondary iron overload-kidney injury.

Topics: Apoptosis; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Cation Transport Proteins; Epithelial Cells; Ferric Compounds; HEK293 Cells; Humans; Iron; Iron Overload; Kidney; Nifedipine; Oxidative Stress; Quaternary Ammonium Compounds; Reactive Oxygen Species; Verapamil

BACKGROUND Iron overload is a prominent characteristic of liver injury, but there is no effective treatment at present. Qizhufang (ZSF) is a Chinese herbal formula showed anti-HBV activities, improved liver function, and anti-fibrosis effect. ZSF showed a series of liver-protection functions, but whether ZSF can relieve hepatic iron overload is still unclear. MATERIAL AND METHODS Ferric ammonium citrate (FAC) was used to construct iron-overloaded LO2 cells. The cell apoptosis and proliferation were measured by flow cytometry and CCK-8 assay, respectively. ROS level was analyzed by fluorescence probe. RNA and protein expressions were assessed by real-time PCR and Western blot. RESULTS FAC upregulated apoptosis rate, ROS level, and expression of hepcidin and p-STAT3, but suppressed proliferation and expression of DMT1, FPN1, and CP in LO2 cells. However, Qizhufang (ZSF) reversed the effect of FAC. We also found that hepcidin overexpression suppressed the expressions of DMT1, FPN1, and CP, which were reversed by ZSF. Additionally, STAT3 inhibitor AG490 suppressed hepcidin expression. Moreover, exogenous IL-6 reversed the effect of ZSF on apoptosis rate, ROS level, and the expression of hepcidin, DMT1, FNP1, CP, and p-STAT3. CONCLUSIONS Qizhufang (ZSF) can ameliorate iron overload-induced injury by suppressing hepcidin via the STAT3 pathway in LO2 cells.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; China; Drugs, Chinese Herbal; Ferric Compounds; Hepcidins; Humans; Interleukin-6; Iron; Iron Overload; Liver; Medicine, Chinese Traditional; Quaternary Ammonium Compounds; Reactive Oxygen Species; Signal Transduction; STAT3 Transcription Factor

Iron overload increases the risk of osteoporosis, which leads to an increase in the incidences of bone fracture after menopause. In vitro studies have demonstrated that excess iron can inhibit osteoblast activity. Hepcidin, a central regulator of iron homeostasis, prevents iron overload, and thus, it is considered to have anti-osteoporosis effects. In this study, a zebrafish model was employed to investigate the therapeutic role of hepcidin in iron overload-induced inhibition of bone formation. Our results show that ferric ammonium citrate (FAC) treatment decreased osteoblast-specific gene expression (runx2a, runx2b, and bglap) and bone mineralization in the zebrafish embryo, accompanied with increased whole-body iron levels and oxidative stress. Bone mineralization and osteoblast-specific gene expression increased with the microinjection of hepcidin-flag Capped-mRNA into zebrafish embryos. Moreover, the whole-body iron content and oxidative stress in the iron-overloaded zebrafish embryos decreased when microinjection of hepcidin preceded the FAC treatment. Therefore, our study suggests that hepcidin could prevent and rescue reduced bone formation caused by FAC treatment by preventing iron absorption.

Topics: Animals; Anti-Infective Agents; Bone Development; Dose-Response Relationship, Drug; Down-Regulation; Ferric Compounds; Gene Expression Regulation; Hepcidins; Iron Overload; Osteoblasts; Quaternary Ammonium Compounds; Zebrafish

Topics: Animals; Autophagy; Cell Line; Disease Models, Animal; Ferric Compounds; Glutathione; Heme Oxygenase-1; Humans; Iron Chelating Agents; Iron Overload; Oxidative Stress; Quaternary Ammonium Compounds; Reactive Oxygen Species; Superoxide Dismutase; Thioctic Acid; Zebrafish

We have previously demonstrated that iron overload in hepatic reticuloendothelial system cells (RES) is associated with severe nonalcoholic steatohepatitis (NASH) and advanced fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). Recruited myeloid-derived macrophages have gained a pivotal position as drivers of NASH progression and fibrosis. In this study, we used bone marrow-derived macrophages (BMDM) from C57Bl6 mice as surrogates for recruited macrophages and examined the effect of iron on macrophage polarization. Treatment with iron (ferric ammonium citrate, FAC) led to increased expression levels of M1 markers: CCL2, CD14, iNOS, IL-1β, IL-6, and TNF-α; it also increased protein levels of CD68, TNF-α, IL-1β, and IL-6 by flow cytometry. This effect could be reversed by desferrioxamine, an iron chelator. Furthermore, iron loading of macrophages in the presence of IL-4 led to the down-regulation of M2 markers: arginase-1, Mgl-1, and M2-specific transcriptional regulator, KLF4. Iron loading of macrophages with IL-4 also resulted in reduced phosphorylation of STAT6, another transcriptional regulator of M2 activation. Dietary iron overload of C57Bl6 mice led to hepatic macrophage M1 activation. Iron overload also stimulated hepatic fibrogenesis. Histologic analysis revealed that iron overload resulted in steatohepatitis. Furthermore, NAFLD patients with hepatic RES iron deposition had increased hepatic gene expression levels of M1 markers, IL-6, IL-1β, and CD40 and reduced gene expression of an M2 marker, TGM2, relative to patients with hepatocellular iron deposition pattern. We conclude that iron disrupts the balance between M1/M2 macrophage polarization and leads to macrophage-driven inflammation and fibrogenesis in NAFLD.

Topics: Adult; Animals; Chemokine CCL2; Deferoxamine; Diet, Fat-Restricted; Female; Femur; Ferric Compounds; Gene Expression Regulation; Humans; Interleukin-1beta; Iron Carbonyl Compounds; Iron Chelating Agents; Iron Overload; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Liver Cirrhosis; Macrophages; Male; Mice; Mice, Inbred C57BL; Middle Aged; Non-alcoholic Fatty Liver Disease; Quaternary Ammonium Compounds; Signal Transduction; STAT6 Transcription Factor; Tibia; Tumor Necrosis Factor-alpha

Iron overload has recently been associated with the changes in the bone microstructure that occur in osteoporosis. However, the effect of iron overload on osteoblasts is unclear. The purpose of this study was to explore the function of divalent metal transporter 1 (DMT1) in the pathological processes of osteoporosis. Osteoblast hFOB1.19 cells were cultured in medium supplemented with different concentrations (0, 50, 100, 200, 300, 400, 500 μmol/L) of ferric ammonium citrate (FAC) as a donor of ferric ions. We used western blotting and immunofluorescence to determine the levels of DMT1 after treatment with FAC. Apoptosis was evaluated by detecting the levels of cleaved caspase 3, BCL2, and BAX with western blotting. Autophagy was evaluated by detecting the levels of LC3 with western blotting and immunofluorescence. Beclin-1 expression was also assessed with western blotting. The autophagy inhibitor 3-methyladenine was used to determine whether autophagy affects the apoptosis induced by FAC. Our results show that FAC increased the levels of DMT1, upregulated the expression of BCL2, and downregulated the apoptosis-related proteins cleaved caspase 3 and BAX. Both LC3I/LC3II levels and beclin-1 were also increased, indicating that FAC increases the accumulation of autophagosomes in hFOB1.19 cells. FAC-induced autophagy was increased by the apoptosis inhibitor 3-MA but was reduced in DMT1 shRNA hFOB1.19 cells. These results suggest that the increased expression of DMT1 induces iron overload and iron overload induces osteoblast autophagy and apoptosis, thus affecting the pathological processes of osteoporosis. Clarifying the mechanisms underlying the effects of DMT1 will allow the identification of novel targets for the prevention and treatment of osteoporosis.

Topics: Apoptosis; Autophagy; bcl-2-Associated X Protein; Caspase 3; Ferric Compounds; Gene Expression Regulation; Humans; Iron; Iron Overload; Osteoblasts; Osteoporosis; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Quaternary Ammonium Compounds; Transcription Factors

Iron overload induces severe damage to several vital organs such as the liver, heart and bone, and thus contributes to the dysfunction of these organs. The aim of this study is to investigate whether iron overload causes the apoptosis and necrosis of bone marrow mesenchymal stem cells (BMSCs) and melatonin may prevent its toxicity. Perls' Prussion blue staining showed that exposure to increased concentrations of ferric ammonium citrate (FAC) induced a gradual increase of intracellular iron level in BMSCs. Trypan blue staining demonstrated that FAC decreased the viability of BMSCs in a concentration-dependent manner. Notably, melatonin protected BMSCs against apoptosis and necrosis induced by FAC and it was vertified by Live/Dead, TUNEL and PI/Hoechst stainings. Furthermore, melatonin pretreatment suppressed FAC-induced reactive oxygen species accumulation. Western blot showed that exposure to FAC resulted in the decrease of anti-apoptotic protein Bcl-2 and the increase of pro-apoptotic protein Bax and Cleaved Caspase-3, and necrosis-related proteins RIP1 and RIP3, which were significantly inhibited by melatonin treatment. At last, melatonin receptor blocker luzindole failed to block the protection of BMSCs apoptosis and necrosis by melatonin. Taken together, melatonin protected BMSCs from iron overload induced apoptosis and necrosis by regulating Bcl-2, Bax, Cleaved Caspase-3, RIP1 and RIP3 pathways.

Topics: Animals; Apoptosis; Bone Marrow Cells; Caspase 3; Cell Survival; Disease Models, Animal; Ferric Compounds; Iron Overload; Male; Melatonin; Mice; Necrosis; Oxidative Stress; Protective Agents; Proto-Oncogene Proteins c-bcl-2; Quaternary Ammonium Compounds; Reactive Oxygen Species; Signal Transduction

Bone marrow mesenchymal stem cells (BMSCs) are an expandable population of stem cells which can differentiate into osteoblasts, chondrocytes and adipocytes. Dysfunction of BMSCs in response to pathological stimuli contributes to bone diseases. Melatonin, a hormone secreted from pineal gland, has been proved to be an important mediator in bone formation and mineralization. The aim of this study was to investigate whether melatonin protected against iron overload-induced dysfunction of BMSCs and its underlying mechanisms. Here, we found that iron overload induced by ferric ammonium citrate (FAC) caused irregularly morphological changes and markedly reduced the viability in BMSCs. Consistently, osteogenic differentiation of BMSCs was significantly inhibited by iron overload, but melatonin treatment rescued osteogenic differentiation of BMSCs. Furthermore, exposure to FAC led to the senescence in BMSCs, which was attenuated by melatonin as well. Meanwhile, melatonin was able to counter the reduction in cell proliferation by iron overload in BMSCs. In addition, protective effects of melatonin on iron overload-induced dysfunction of BMSCs were abolished by its inhibitor luzindole. Also, melatonin protected BMSCs against iron overload-induced ROS accumulation and membrane potential depolarization. Further study uncovered that melatonin inhibited the upregulation of p53, ERK and p38 protein expressions in BMSCs with iron overload. Collectively, melatonin plays a protective role in iron overload-induced osteogenic differentiation dysfunction and senescence through blocking ROS accumulation and p53/ERK/p38 activation.

Topics: Adipogenesis; Animals; Bone Marrow Cells; Cell Differentiation; Cell Proliferation; Cellular Senescence; Ferric Compounds; Iron Overload; Iron-Dextran Complex; Male; MAP Kinase Signaling System; Melatonin; Mesenchymal Stem Cells; Mice, Inbred C57BL; Mitochondria; Osteogenesis; Quaternary Ammonium Compounds; Reactive Oxygen Species; Tryptamines

Iron overload leads to multiple organ damage including endocrine organ dysfunctions. Hypogonadism is the most common non-diabetic endocrinopathy in primary and secondary iron overload syndromes.. To explore the molecular determinants of iron overload-induced hypogonadism with specific focus on hypothalamic derangements. A dysmetabolic male murine model fed iron-enriched diet (IED) and cell-based models of gonadotropin-releasing hormone (GnRH) neurons were used.. Mice fed IED showed severe hypogonadism with a significant reduction of serum levels of testosterone (-83%) and of luteinizing hormone (-86%), as well as reduced body weight gain, body fat and plasma leptin. IED mice had a significant increment in iron concentration in testes and in the pituitary. Even if iron challenge of in vitro neuronal models (GN-11 and GT1-7 GnRH cells) resulted in 10- and 5-fold iron content increments, respectively, no iron content changes were found in vivo in hypothalamus of IED mice. Conversely, mice placed on IED showed a significant increment in hypothalamic GnRH gene expression (+34%) and in the intensity of GnRH-neuron innervation of the median eminence (+1.5-fold); similar changes were found in the murine model HFE. IED-fed adult male mice show severe impairment of hypothalamus-pituitary-gonadal axis without a relevant contribution of the hypothalamic compartment, which thus appears sufficiently protected from systemic iron overload.

Topics: Animals; Cell Line; Diet; Ferric Compounds; Gonadotropin-Releasing Hormone; Homeostasis; Hypogonadism; Hypothalamo-Hypophyseal System; Hypothalamus; Iron; Iron Overload; Male; Mice, Inbred C57BL; Phenotype; Quaternary Ammonium Compounds; Testis

Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe

Topics: 2,2'-Dipyridyl; Acinetobacter baumannii; Acinetobacter Infections; Anemia, Iron-Deficiency; Animals; Cation Transport Proteins; Cations, Divalent; Disease Models, Animal; Ferric Compounds; Firefly Luciferin; Fluorescent Dyes; Gene Expression Regulation; Hepcidins; Homeostasis; Iron; Iron Overload; Iron Regulatory Protein 1; Iron Regulatory Protein 2; Luminescent Measurements; Mice; Mice, Transgenic; Quaternary Ammonium Compounds; Receptors, Transferrin; Signal Transduction; Transferrin

Topics: Animals; Antioxidants; Carcinogens; Cells, Cultured; Curcuma; Curcumin; Epithelial Cells; Ferric Compounds; Iron; Iron Overload; Liver; Liver Neoplasms; Oxidative Stress; Plant Extracts; Quaternary Ammonium Compounds; Rats

To establish macrophage iron overload model in vitro by co-culture macrophages with iron, and to explore the effect of iron overload on cell reactive oxygen species (ROS) and the impact of ROS on macrophages.. Iron overload group were treated with different concentrations (0, 5, 10, 20, 40, 80 μmol/L respectively) of ferric ammonium citrate (FAC). The control group was the group of macrophages without FAC treatment. We detected the number and state of cells, metabolic activity, the change of phagocytosis, the levels of ROS and reactive nitrogen, and changes of related oxidative stress signaling pathways in different groups. Changes in the above indexes were detected after application of deferasirox (DFX) to remove iron and the antioxidant N -acetylcysteine (NAC) to clear excess oxidative stress.. (1)The levels of labile iron pool (LIP) in macrophages co-cultivated with iron was increased with the increase of iron concentration in a dose-dependent manner. The LIP levels was the highest in the macrophages treated with 80 μmol/L. (2)The increase of FAC concentration, the metabolic activity of macrophages in the 5 FAC-treated groups decreased to 51.58%, 40.98%, 16.23%, 3.46%, and 0.05% of the activity level of the control group (all P< 0.05). The group with the metabolic activity decreased to 16.23% (20 μmol/L) was selected as the iron overload group for the following experiments. (3)Compared with the control group, the number of macrophages in the iron overload group reduced to 32.80% (P<0.05), and the state of cells changed from adherence to partial suspension. The phagocytosis of macrophages in the iron overload group reduced to 20.40% of the control group (P<0.05). (4)Our further experiment showed that the levels of ROS and the activity nitrogen in the iron overload group increased by 7.71-and 1.45-fold compared with the control group (both P<0.05). The RT-PCR showed up-regulated mRNA expression of genes related with ROS production, i. e. nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX 4) gene related with ROS production and inducible nitric oxide synthase (iNOS) gene related with reactive nitrogen production, down-regulated mRNA expression of glutathione peroxidase 1 (GPX1) gene which participated in ROS clearance. Moreover, mRNA expression of phosphatidylinositol-3-kinase (PI3K) gene involved in oxidative stress signaling pathway in the iron overload group was up-regulated, while fork head protein O3 (FOXO3) which regulated oxidative stress through negative feedback showed a down-regulation level of mRNA expression compared with the control group. (5)After iron chelation and antioxidant treatment, the above-mentioned damage in the iron overload group were partially reversed.. The damages of iron overload on macrophages may be mediated by inducing oxidative stress and activating oxidative stress signaling pathways. Our established model provides a method to explore the mechanism of iron overload on macrophage, and may shed some new light on possible therapeutic target in treating iron overload patients.

Topics: Acetylcysteine; Antioxidants; Down-Regulation; Ferric Compounds; Humans; Iron; Iron Overload; Macrophages; Oxidative Stress; Phosphatidylinositol 3-Kinases; Quaternary Ammonium Compounds; Reactive Oxygen Species; Signal Transduction

Iron is necessary for neuronal functions; however, excessive iron accumulation caused by impairment of iron balance could damage neurons. Neuronal iron accumulation has been observed in several neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. Nevertheless, the precise mechanisms underlying iron toxicity in neuron cells are not fully understood. In this study, we investigated the mechanism underlying iron overload-induced mitochondrial fragmentation in HT-22 hippocampal neuron cells that were incubated with ferric ammonium citrate (FAC). Mitochondrial fragmentation via dephosphorylation of Drp1 (Ser637) and increased apoptotic neuronal death were observed in FAC-stimulated HT-22 cells. Furthermore, the levels of intracellular calcium (Ca(2+)) were increased by iron overload. Notably, chelation of intracellular Ca(2+) rescued mitochondrial fragmentation and neuronal cell death. In addition, iron overload activated calcineurin through the Ca(2+)/calmodulin and Ca(2+)/calpain pathways. Pretreatment with the calmodulin inhibitor W13 and the calpain inhibitor ALLN attenuated iron overload-induced mitochondrial fragmentation and neuronal cell death. Therefore, these findings suggest that Ca(2+)-mediated calcineurin signals are a key player in iron-induced neurotoxicity by regulating mitochondrial dynamics. We believe that our results may contribute to the development of novel therapies for iron toxicity related neurodegenerative disorders.

Topics: Animals; Calcineurin; Calcium; Calcium Signaling; Calmodulin; Calpain; Cell Death; Cell Line; Cell Survival; Dynamins; Ferric Compounds; Hippocampus; Iron; Iron Overload; Leupeptins; Mice; Mitochondria; Neurons; Quaternary Ammonium Compounds; Sulfonamides

Iron dyshomeostasis is one of the primary causes of neuronal death in Alzheimer's disease (AD). Huperzine A (HupA), a natural inhibitor of acetylcholinesterase (AChE), is a licensed anti-AD drug in China and a nutraceutical in the United Sates. Here, we investigated the protective effects of HupA against iron overload-induced injury in neurons.. Rat cortical neurons were treated with ferric ammonium citrate (FAC), and cell viability was assessed with MTT assays. Reactive oxygen species (ROS) assays and adenosine triphosphate (ATP) assays were performed to assess mitochondrial function. The labile iron pool (LIP) level, cytosolic-aconitase (c-aconitase) activity and iron uptake protein expression were measured to determine iron metabolism changes. The modified Ellman's method was used to evaluate AChE activity.. HupA significantly attenuated the iron overload-induced decrease in neuronal cell viability. This neuroprotective effect of HupA occurred concurrently with a decrease in ROS and an increase in ATP. Moreover, HupA treatment significantly blocked the upregulation of the LIP level and other aberrant iron metabolism changes induced by iron overload. Additionally, another specific AChE inhibitor, donepezil (Don), at a concentration that caused AChE inhibition equivalent to that of HupA negatively, influenced the aberrant changes in ROS, ATP or LIP that were induced by excessive iron.. We provide the first demonstration of the protective effects of HupA against iron overload-induced neuronal damage. This beneficial role of HupA may be attributed to its attenuation of oxidative stress and mitochondrial dysfunction and elevation of LIP, and these effects are not associated with its AChE-inhibiting effect.

Topics: Acetylcholinesterase; Adenosine Triphosphate; Alkaloids; Animals; Antioxidants; Cells, Cultured; Cerebral Cortex; Ferric Compounds; Iron; Iron Overload; Mitochondria; Neurons; Oxidative Stress; Quaternary Ammonium Compounds; Rats, Sprague-Dawley; Reactive Oxygen Species; Sesquiterpenes; Signal Transduction

Postmenopausal osteoporosis is a metabolic disease associated with estrogen deficiency. The results of numerous studies have revealed the positive correlation between iron accumulation and postmenopausal osteoporotic status. Although the results of previous studies have indicated that estrogen or iron alone have an effect on bone metabolism, their combined effects are not well defined. Using an in vivo mouse model, we found that bone mass was minimally affected by an excess of iron in the presence of estrogen. Once the source of estrogen was removed (ovariectomy), iron accumulation significantly decreased bone mass. These effects were accompanied by fluctuations in the level of oxidative stress. To determine whether these effects were related to bone formation or bone resorption, primary osteoblasts (OBs), RAW264.7 cells, and bone-marrow-derived macrophages were used for in vitro experiments. We found that iron accumulation did inhibit the activity of OBs. However, estrogen had little effect on this inhibition. In contrast, iron promoted osteoclast differentiation through the production of reactive oxygen species. Estrogen, a powerful reactive oxygen scavenger, suppressed this effect in osteoclasts. Our data provided direct evidence that iron affected the bone mass only in the absence of estrogen. The inhibitory effect of estrogen on iron-induced osteopenia was particularly relevant to bone resorption rather than bone formation.

Topics: Animals; Bone and Bones; Bone Density; Bone Resorption; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Estradiol; Female; Ferric Compounds; Iron Overload; Macrophages; Mice; Osteoblasts; Osteoclasts; Ovariectomy; Oxidative Stress; Quaternary Ammonium Compounds; Reactive Oxygen Species

To investigate whether the treatment of oxidized low density lipoprotein (ox-LDL) could reduce ceruloplasmin (Cp) expression and then, influence iron efflux in macrophages.. RAW264.7 cells were treated by lipopolysaccharides (LPS), ferric ammonium citrate (FAC), deferoxamine (DFO) and ox-LDL. Biochemical analysis and histological assays were performed to detect the iron-related protein including ferritin (Ft), transferrin receptor (TfR), ferroportin 1 (FPN1) and Cp. And the cholesterol enzyme was used to evaluate cells' cholesterol content.. Western blot showed that protein level of FPN1, Ft increased and protein level of TfR decreased after the treatment of FAC. The mRNA level (t=-5.995, P=0.019) and the oxidative activity (t=-9.875, P=0.001) of Cp increased significantly after the treatment of LPS+FAC compared to control group. The treatment of ox-LDL could reduce protein level of Cp compared to LPS treated cells and no different protein levels of Ft and Ferroportin was found after the treatment of ox-LDL as shown by Western blot. The protein level, ferroxidase activities (t=6.663, P=0.003) and mRNA levels (t=8.948, P=0.001) of Cp in LPS+ox-LDL+FAC group were reduced than LPS+FAC group. The protein levels of Ft and FPN1 in LPS+ox-LDL+FAC group was significant higher than LPS+FAC group as shown by Western blot.. The findings suggest that the expression of Cp is reduced in foam cells and leads to obstructed iron efflux in iron overloaded foam cells.

Topics: Animals; Antigens, CD; Cation Transport Proteins; Ceruloplasmin; Cholesterol; Ferric Compounds; Ferritins; Foam Cells; Iron; Iron Overload; Lipids; Lipoproteins, LDL; Macrophages; Mice; Oxidation-Reduction; Quaternary Ammonium Compounds; RAW 264.7 Cells; Receptors, Transferrin

Ticks are obligate hematophagous parasites that have successfully developed counteractive means against their hosts' immune and hemostatic mechanisms, but their ability to cope with potentially toxic molecules in the blood remains unclear. Iron is important in various physiological processes but can be toxic to living cells when in excess. We previously reported that the hard tick Haemaphysalis longicornis has an intracellular (HlFER1) and a secretory (HlFER2) ferritin, and both are crucial in successful blood feeding and reproduction. Ferritin gene silencing by RNA interference caused reduced feeding capacity, low body weight and high mortality after blood meal, decreased fecundity and morphological abnormalities in the midgut cells. Similar findings were also previously reported after silencing of ferritin genes in another hard tick, Ixodes ricinus. Here we demonstrated the role of ferritin in protecting the hard ticks from oxidative stress. Evaluation of oxidative stress in Hlfer-silenced ticks was performed after blood feeding or injection of ferric ammonium citrate (FAC) through detection of the lipid peroxidation product, malondialdehyde (MDA) and protein oxidation product, protein carbonyl. FAC injection in Hlfer-silenced ticks resulted in high mortality. Higher levels of MDA and protein carbonyl were detected in Hlfer-silenced ticks compared to Luciferase-injected (control) ticks both after blood feeding and FAC injection. Ferric iron accumulation demonstrated by increased staining on native HlFER was observed from 72 h after iron injection in both the whole tick and the midgut. Furthermore, weak iron staining was observed after Hlfer knockdown. Taken together, these results show that tick ferritins are crucial antioxidant molecules that protect the hard tick from iron-mediated oxidative stress during blood feeding.

Topics: Animals; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Female; Ferric Compounds; Ferritins; Iron Overload; Ixodidae; Malondialdehyde; Oxidative Stress; Protein Carbonylation; Quaternary Ammonium Compounds; RNA Interference; Statistics, Nonparametric; Time Factors

Iron overload is associated with increased severity of nonalcoholic fatty liver disease (NAFLD) including progression to nonalcoholic steatohepatitis and hepatocellular carcinoma.. To identify potential role(s) of iron in NAFLD, we measured its effects on pathways of oxidative stress and insulin signaling in AML-12 mouse hepatocytes.. Rapid iron overload was induced with 50 μM ferric ammonium citrate and 8-hydroxyquinoline. Insulin response was measured by Western blot of phospho-protein kinase B. Lipid content was determined by staining with Oil Red O. Reactive oxygen species (ROS) were measured by flow cytometry using 5-(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Oxidative stress was measured by Western blots for phospho-jnk and phospho-p38.. Iron increased ROS (p < 0.001) and oxidative stress (p < 0.001) and decreased insulin signaling by 33 % (p < 0.001). Treatment with stearic or oleic acids (200 μM) increased cellular lipid content and differentially modulated effects of iron. Stearic acid potentiated iron-induced ROS levels by two-fold (p < 0.05) and further decreased insulin response 59 % (p < 0.05) versus iron alone. In contrast, cells treated with oleic acid were protected against iron-mediated injury; ROS levels were decreased by half (p < 0.01) versus iron alone while insulin response was restored to control (untreated) levels. The anti-oxidant curcumin reduced effects of iron on insulin signaling, ROS, and oxidative stress (p < 0.01). Curcumin was similarly effective in cells treated with both stearic acid and iron.. An in vitro model of NAFLD progression is described in which iron-induced oxidative stress inhibits insulin signaling. Pathophysiological effects of iron were increased by saturated fat and decreased by curcumin.

Topics: Animals; Biomarkers; Blotting, Western; Cell Line; Fatty Acids, Nonesterified; Fatty Liver; Ferric Compounds; Flow Cytometry; Hepatocytes; Insulin; Insulin Resistance; Iron Overload; Mice; Non-alcoholic Fatty Liver Disease; Oxidative Stress; Oxyquinoline; Quaternary Ammonium Compounds; Reactive Oxygen Species

Iron-overload cardiomyopathy is a major cause of death in thalassemic patients. However, pathways of non-transferrin-bound iron (NTBI) uptake into cardiomyocytes under iron-overload conditions are still controversial. We previously demonstrated that Fe(2+) uptake in thalassemic cardiomyocytes is mainly mediated by T-type calcium channels (TTCCs). However, direct evidence regarding Fe(3+) uptake, the other form of NTBI, in thalassemic cardiomyocytes has never been investigated. Hearts from genetic-altered β-thalassemic mice and adult wild-type (WT) mice were used for cultured ventricular cardiomyocytes. Blockers for L-type calcium channel (LTCC), TTCC, transferrin receptor1 (TfR1), and divalent metal transporter1 (DMT1) were used, and quantification of cellular iron uptake was performed by the acetoxymethyl ester of calcein fluorescence assay. Cellular uptake of Fe(3+) under iron-overload conditions in cultured ventricular myocytes of thalassemic mice was greater than that of WT cells (P < 0.01). The iron chelator, deferoxamine, could prevent Fe(3+) uptake into cultured cardiomyocytes. However, blockers of TfR1, DMT1, LTCC, and TTCC could not prevent Fe(3+) uptake into cardiomyocytes. Our findings indicated that, unlike Fe(2+), Fe(3+) uptake in cultured thalassemic cardiomyocytes is not mainly mediated by TfR1, DMT1, LTCC, and TTCC, suggesting that another alternative pathway could play a major role in Fe(3+) uptake in thalassemic cardiomyocytes.

Topics: Animals; Azoles; beta-Thalassemia; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Calcium Channels, T-Type; Cation Transport Proteins; Cell Survival; Cells, Cultured; Deferoxamine; Dihydropyridines; Disease Models, Animal; Ferric Compounds; Heart Ventricles; Iron Overload; Isoindoles; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Cardiac; Nitrophenols; Organophosphorus Compounds; Organoselenium Compounds; Quaternary Ammonium Compounds; Receptors, Transferrin; Verapamil

Although iron overload induces oxidative stress and brain mitochondrial dysfunction, and is associated with neurodegenerative diseases, brain mitochondrial iron uptake has not been investigated. We determined the role of mitochondrial calcium uniporter (MCU) in brain mitochondria as a major route for iron entry. We hypothesized that iron overload causes brain mitochondrial dysfunction, and that the MCU blocker prevents iron entry into mitochondria, thus attenuating mitochondrial dysfunction.. Isolated brain mitochondria from male Wistar rats were used. Iron (Fe(2+) and Fe(3+)) at 0-286 μM were applied onto mitochondria at various incubation times (5-30 min), and the mitochondrial function was determined. Effects of MCU blocker (Ru-360) and iron chelator were studied.. Both Fe(2+) and Fe(3+) entered brain mitochondria and caused mitochondrial swelling in a dose- and time-dependent manner, and caused mitochondrial depolarization and increased ROS production. However, Fe(2+) caused more severe mitochondrial dysfunction than Fe(3+). Although all drugs attenuated mitochondrial dysfunction caused by iron overload, only an MCU blocker could completely prevent ROS production and mitochondrial depolarization.. Our findings indicated that iron overload caused brain mitochondrial dysfunction, and that an MCU blocker effectively prevented this impairment, suggesting that MCU could be the major portal for brain mitochondrial iron uptake.

Topics: Animals; Brain; Calcium Channels; Citrates; Dose-Response Relationship, Drug; Ferric Compounds; Ferrous Compounds; In Vitro Techniques; Iron Chelating Agents; Iron Overload; Male; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Mitochondria; Mitochondrial Swelling; Quaternary Ammonium Compounds; Rats; Rats, Wistar; Reactive Oxygen Species; Ruthenium Compounds; Time Factors

In order to understand mechanisms involved in osteoporosis observed during iron overload diseases, we analyzed the impact of iron on a human osteoblast-like cell line. Iron exposure decreases osteoblast phenotype. HHIPL-2 is an iron-modulated gene which could contribute to these alterations. Our results suggest osteoblast impairment in iron-related osteoporosis.. Iron overload may cause osteoporosis. An iron-related decrease in osteoblast activity has been suggested.. We investigated the effect of iron exposure on human osteoblast cells (MG-63) by analyzing the impact of ferric ammonium citrate (FAC) and iron citrate (FeCi) on the expression of genes involved in iron metabolism or associated with osteoblast phenotype. A transcriptomic analysis was performed to identify iron-modulated genes.. FAC and FeCi exposure modulated cellular iron status with a decrease in TFRC mRNA level and an increase in intracellular ferritin level. FAC increased ROS level and caspase 3 activity. Ferroportin, HFE and TFR2 mRNAs were expressed in MG-63 cells under basal conditions. The level of ferroportin mRNA was increased by iron, whereas HFE mRNA level was decreased. The level of mRNA alpha 1 collagen type I chain, osteocalcin and the transcriptional factor RUNX2 were decreased by iron. Transcriptomic analysis revealed that the mRNA level of HedgeHog Interacting Protein Like-2 (HHIPL-2) gene, encoding an inhibitor of the hedgehog signaling pathway, was decreased in the presence of FAC. Specific inhibition of HHIPL-2 expression decreased osteoblast marker mRNA levels. Purmorphamine, hedgehog pathway activator, increased the mRNA level of GLI1, a target gene for the hedgehog pathway, and decreased osteoblast marker levels. GLI1 mRNA level was increased under iron exposure.. We showed that in human MG-63 cells, iron exposure impacts iron metabolism and osteoblast gene expression. HHIPL-2 gene expression modulation may contribute to these alterations. Our results support a role of osteoblast impairment in iron-related osteoporosis.

Topics: Cation Transport Proteins; Cells, Cultured; Citric Acid; Ferric Compounds; Ferrous Compounds; Gene Expression Regulation; Hemochromatosis Protein; Histocompatibility Antigens Class I; Humans; Iron Overload; Membrane Proteins; Osteoblasts; Oxidative Stress; Phenotype; Quaternary Ammonium Compounds

Bone metabolism has a close relationship with iron homeostasis. To examine the effects of iron excess and iron deficiency on the biological activities of osteoblast in vitro, human osteoblast cells (hFOB1.19) were incubated in a medium supplemented with 0-200 μmol/L ferric ammonium citrate and 0-20 μmol/L deferoxamine. The intracellular iron was measured by a confocal laser scanning microscope. Proliferation of osteoblasts was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptotic cells were detected using annexin intervention V/PI staining with a flow cytometry. Alkaline phosphatase (ALP) activity was measured using an ALP assay kit. The number of calcified nodules and mineral area was evaluated by von Kossa staining assay. The expressions of type I collagen and osteocalcin of cultured osteoblasts were detected by reverse transcriptase polymerase chain reaction and Western blot. Intracellular reactive oxygen species (ROS) was measured using the oxidation-sensitive dye 2,7-dichlorofluorescin diacetate by flow cytometry. The results indicated that excessive iron inhibited osteoblast activity in a concentration-dependent manner. Low iron concentrations, in contrast, produced a biphasic manner on osteoblasts: mild low iron promoted osteoblast activity, but serious low iron inhibited osteoblast activity. Osteogenesis was optimal in certain iron concentrations. The mechanism underlying biological activity invoked by excessive iron may be attributed to increased intracellular ROS levels.

Topics: Alkaline Phosphatase; Anemia, Iron-Deficiency; Apoptosis; Calcification, Physiologic; Cell Line; Cell Proliferation; Collagen Type I; Deferoxamine; Dietary Supplements; Ferric Compounds; Gene Expression Regulation; Hormesis; Humans; Iron; Iron Deficiencies; Iron Overload; Osteoblasts; Osteocalcin; Osteogenesis; Quaternary Ammonium Compounds; Reactive Oxygen Species; Siderophores

Excessive neuronal iron has been proposed to contribute to the pathology of several neurodegenerative diseases including Alzheimer's and Parkinson's diseases. This work characterized human neuroblastoma IMR-32 cells exposure to ferric ammonium citrate (FAC) as a model of neuronal iron overload and neurodegeneration. The consequences of FAC treatment on neuronal oxidative stress and on the modulation of the oxidant-sensitive transcription factors AP-1 and NF-κB were investigated. Incubation with FAC (150μM) resulted in a time (3-72h)-dependent increase in cellular iron content, and was associated with cell oxidant increase. FAC caused a time-dependent (3-48h) increase in nuclear AP-1- and NF-κB-DNA binding. This was associated with the upstream activation of the mitogen activated kinases ERK1/2, p38 and JNK and of IκBα phosphorylation and degradation. After 72h incubation with FAC, cell viability was 40% lower than in controls. Iron overload caused apoptotic cell death. After 48-72h of incubation with FAC, caspase 3 activity was increased, and chromatin condensation and nuclear fragmentation were observed. In summary, the exposure of IMR-32 cells to FAC is associated with increased oxidant cell levels, activation of redox-sensitive signals, and apoptosis.

Topics: Cell Line, Tumor; Cell Survival; Ferric Compounds; Humans; Iron Overload; Neuroblastoma; Oxidation-Reduction; Quaternary Ammonium Compounds; Signal Transduction

The recovery from iron overload is hampered by the limited number of pathways and therapeutic agents available for the augmentation of iron secretion/excretion. The present study was aimed to investigate the process of iron storage and release by cultured human hepatoma cells, the role of transferrin receptors and ferritin in this process as well as the effect of iron chelators.. We followed the acquisition, storage and release of iron by cultured cells HepG2 and Hep3B by biochemical means and electron microscopy.. The uptake of iron from diferric transferrin (Trf) was extremely low, while iron as ferric-ammonium-citrate (FAC) was taken up readily, especially by Hep3B cells. Up to 80% of the iron taken up by hepatoma cells was released to the medium. The rate of spontaneous iron release depended on the extent of iron loading. ApoTrf and deferoxamine facilitated release after 1- and 7-day iron-exposure. Up to a third of the radio-iron released from the cells was associated with ferritin. The release of ferritin-iron was not enhanced by either deferoxamine or Trf.. Ferritin-iron release appeared to be an important mechanism of iron discarding in cultured human hepatoma cells, independent of the activity of chelating agents.

Topics: Apoproteins; Carcinoma, Hepatocellular; Culture Media; Deferoxamine; Ferric Compounds; Ferritins; Humans; Iron; Iron Chelating Agents; Iron Overload; Liver Neoplasms; Microscopy, Electron; Quaternary Ammonium Compounds; Receptors, Transferrin; Transferrin; Tumor Cells, Cultured

The liver is one of the principal sites of iron overload in diseases such as hemochromatosis and beta thalassemia. Hence, much effort has been invested in examining the mechanisms of Fe uptake by hepatocytes. In the present study we have examined the effect of small molecular weight (M(r)) Fe complexes on Fe uptake from iron 59-labeled transferrin (Tf) and 59Fe-labeled citrate by primary cultures of hepatocytes. This was important to assess because Fe-citrate and saturated diferric Tf coexist in the serum of patients with untreated Fe overload. Preincubation of hepatocytes with the low-M(r) Fe complex ferric ammonium citrate (FAC; 25 microg/mL; (Fe) = 4.4 microg/mL) followed by incubation with 59Fe-Tf or 59Fe-citrate ((Fe) = 0.25 to 25 micromol/L) resulted in the marked stimulation of 59Fe uptake. For example, at a physiologically relevant Tf-Fe concentration of 25 micromol/L, there was an 8-fold increase in 59Fe uptake by cells incubated with FAC compared to control cells. In contrast, at Tf-Fe concentrations of 0.25 to 2.5 micromol/L, 59Fe uptake in FAC-treated cells was only 1-fold to 3-fold greater than that in the corresponding controls. These data suggest that the FAC-activated Fe uptake process predominates at physiologically relevant Tf concentrations above the saturation of the Tf receptor (TfR). This is the first study to demonstrate that preincubation of hepatocytes with Iow-M(r)Fe complexes can markedly increase Fe uptake from diferric Tf. In conclusion, these results may help to explain the loading of hepatocytes with Fe that occurs in Fe-overload disease despite marked down-regulation of the TfR.

Topics: Animals; Cells, Cultured; Ferric Compounds; Iron; Iron Overload; Iron Radioisotopes; Liver; Male; Molecular Weight; Quaternary Ammonium Compounds; Rats; Rats, Wistar; Transferrin

Iron overload in BALB/c mice by treatment with ferric ammonium citrate promotes the hepatic development of Plasmodium yoelii in vivo and in vitro. This was the result of increased penetration of the parasite into hepatocytes since no effect was observed on parasite transformation or maturation. These results could explain why in endemic regions iron supplementation led, in certain studies, to an increase in clinical episodes of malaria and in the prevalence of malaria infection.

Topics: Animals; Ferric Compounds; Iron; Iron Overload; Liver; Malaria; Male; Mice; Mice, Inbred BALB C; Plasmodium yoelii; Quaternary Ammonium Compounds