2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes.     

Sulfide increases labile iron pool in RD4 cells

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

Transient increase of the labile iron pool in HepG2 cells by intravenous iron preparations.

Intravenous iron, used for the treatment of anemia in chronic renal failure and other diseases, represents a possible source of free iron in tissue cells, particularly in the liver. In this study we examined the effect of different sources of intravenous iron (IVI) on the labile iron pool (LIP) which represents the nonferritin-bound, redox-active iron that is implicated in oxidative stress and cell injury. Furthermore, we examined the role of the LIP for the synthesis of ferritin. We used HepG2 cells as a well known model for hepatoma cells and monitored the LIP with the metal-sensitive fluorescent probe, calcein-AM, the fluorescence of which is quenched on binding to iron. We showed that steady state LIP levels in HepG2 cells were increased transiently, up to three-fold compared to control cells, as an adaptive response to long-term IVI exposure. In relation to the amount of iron in the LIP, the ferritin levels increased and the iron content of ferritin decreased. As any fluctuation in the LIP, even when it is only transient (e.g. after exposure to intravenous iron in this study), may result either in impairment of synthesis of iron containing proteins or in cell injury by pro-oxidants. Such findings in nonreticuloendothelial cells may have important implications in the generation of the adverse effects of chronic iron exposure reported in dialysis patients.     

Red blood cells upregulate cytoprotective proteins and the labile iron pool in dividing human T cells despite a reduction in oxidative stress

We have recently reported that red blood cells (RBC) promote T cell growth and survival by inhibiting activation-induced T cell death. In the present study, we have examined parameters of oxidative stress and intracellular iron in activated T cells and correlated these data with the expression of ferritin, heme oxygenase-1 (HO-1), and the transferrin receptor CD71. T cells growing in the presence of RBC had reduced levels of reactive oxygen species (ROS) and oxidatively modified proteins, suggesting that RBC efficiently counteracted ROS production on the activated T cells. Flow cytometry and immunodetection demonstrated that T cells dividing in the presence of RBC had increased levels of intracellular ferritin rich in L-subunits and HO-1 along with a downmodulation in CD71 expression. Finally, using the fluorescent iron indicator calcein and flow cytometry analysis, we were able to show that a relative amount of the labile iron pool (LIP) was upregulated in T cells growing in the presence of RBC. These findings are consistent with a typical response to iron overload. However, neither heme compounds nor ferric iron reproduced the levels of expansion and survival of T cells induced by intact RBC. Altogether, these data suggest that RBC inhibit apoptosis of activated T cells by a combination of ROS scavenging and upregulation of cytoprotective proteins such as ferritin and HO-1, which may counteract a possible toxic effect of the increased intracellular free iron.     

Detection of iron-containing proteins contributing to the cellular labile iron pool by a native electrophoresis metal blotting technique

The labile iron pool (LIP) plays a role in generation of free radicals and is thus the target of chelators used for the treatment of iron overload. We have previously shown that the LIP is bound mostly to high molecular weight carriers (MW>5000). However, the iron does not remain associated with these proteins during native gel electrophoresis. In this study we describe a new method to reconstruct the interaction of iron with iron-binding proteins. Proteins were separated by native gradient polyacrylamide gel electrophoresis and transfered to polyvinilidene difluoride membrane under native conditions. The immobilized iron-binding proteins are then labeled by 59Fe using a 'titrational blotting' technique and visualized by storage phosphorimaging. At least six proteins, in addition to ferritin and transferrin, are specifically labeled in cellular lysates of human erythroleukemic cells. This technique enables separation and detection of iron-binding proteins or other metal-protein complexes under near-physiological conditions and facilitates identification of weak iron-protein complexes. Using a new native metal blotting method, we have confirmed that specific high molecular weight proteins bind the labile iron pool.     

Mammalian siderophores, siderophore-binding lipocalins, and the labile iron pool

Bacteria use tight-binding, ferric-specific chelators called siderophores to acquire iron from the environment and from the host during infection; animals use proteins such as transferrin and ferritin to transport and store iron. Recently, candidate compounds that could serve endogenously as mammalian siderophore equivalents have been identified and characterized through associations with siderocalin, the only mammalian siderophore-binding protein currently known. Siderocalin, an antibacterial protein, acts by sequestering iron away from infecting bacteria as siderophore complexes. Candidate endogenous siderophores include compounds that only effectively transport iron as ternary complexes with siderocalin, explaining pleiotropic activities in normal cellular processes and specific disease states.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptosis by disruption of intracellular calcium homeostasis in human neuronal cell line SHSY5Y

The persistent xenobiotic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces neurotoxic effects that alters neurodevelopment and behavior both during development and adulthood. There are many ongoing efforts to determine the molecular mechanisms of TCDD-mediated neurotoxicity, the signaling pathways involved and its molecular targets in neurons. In this work, we have used SHSY5Y human neuroblastoma cells to characterize the TCDD-induced toxicity. TCDD produces a loss of viability linked to an increased caspase-3 activity, PARP-1 fragmentation, DNA laddering, nuclear fragmentation and hypodiploid (apoptotic) DNA content, in a similar way than staurosporine, a prototypical molecule of apoptosis induction. In addition, TCDD produces a decrease of mitochondrial membrane potential and an increase of intracellular calcium concentration (P?<?0.05). Finally, based on the high lipophilic properties of the dioxin, we test the TCDD effect on the membrane integrity using sarcoplasmic reticulum vesicles as a model. TCDD produces calcium efflux through the membrane and an anisotropy decrease (P?<?0.05) that reflects an increase in membrane fluidity. Altogether these results support the hypothesis that TCDD toxicity in SHSY5Y neuroblastoma cells provokes the disruption of calcium homeostasis, probably affecting membrane structural integrity, leading to an apoptotic process.     

Overexpression of the ferritin iron-responsive element decreases the labile iron pool and abolishes the regulation of iron absorption by intestinal epithelial (Caco-2) cells

Mammalian cells regulate iron levels tightly through the activity of iron-regulatory proteins (IRPs) that bind to RNA motifs called iron-responsive elements (IREs). When cells become iron-depleted, IRPs bind to IREs present in the mRNAs of ferritin and the transferrin receptor, resulting in diminished translation of the ferritin mRNA and increased translation of the transferrin receptor mRNA. Likewise, intestinal epithelial cells regulate iron absorption by a process that also depends on the intracellular levels of iron. Although intestinal epithelial cells have an active IRE/IRP system, it has not been proven that this system is involved in the regulation of iron absorption in these cells. In this study, we characterized the effect of overexpression of the ferritin IRE on iron absorption by Caco-2 cells, a model of intestinal epithelial cells. Cells overexpressing ferritin IRE had increased levels of ferritin, whereas the levels of the transferrin receptor were decreased. Iron absorption in IRE-transfected cells was deregulated: iron uptake from the apical medium was increased, but the capacity to retain this newly incorporated iron diminished. Cells overexpressing IRE were not able to control iron absorption as a function of intracellular iron, because both iron-deficient cells as well as iron-loaded cells absorbed similarly high levels of iron. The labile iron pool of IRE-transfected cell was extremely low. Likewise, the reduction of the labile iron pool in control cells resulted in cells having increased iron absorption. These results indicate that cells overexpressing IRE do not regulate iron absorption, an effect associated with decreased levels of the regulatory iron pool.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin: examination of biochemical effects involved in the proliferation and differentiation of XB cells

XB, a cell line derived from a mouse teratoma, differentiates into stratified squamous epithelium when incubated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To examine the possible biochemical mediators of this response, we compared the effects produced by TCDD to those elicited by other compounds which stimulate epidermal proliferation and/or differentiation in mice. XB/3T3 cultures keratinize when incubated with cholera toxin, epidermal growth factor (EGF), or TCDD, but not 12-0-tetradecanoylphorbol-13-acetate (TPA). Incubation of XB cells with TCDD (10(-9)M) for 48 hours produces a 20% increase in thymidine incorporation, a response which is neither as large nor as rapid as that produced by cholera toxin, TPA, or EGF. Although both cholera toxin and TCDD stimulate differentiation and thymidine incorporation in XB/3T3 cultures, cholera toxin increases cAMP 30-fold in these cells, while TCDD does not affect cAMP accumulation at any of the times studies (15 min to 120 hours). Inhibitors of arachidonic acid metabolism, which block epidermal proliferative responses to TPA in vivo, do not prevent the differentiation of XB cells in response to TCDD. In XB/3T3 cultures, TPA stimulates arachidonic acid release at all times tested (1,6, and 24 hours) and increases the incorporation of 32Pi into total phospholipids and phosphatidylcholine after 3 hours. In contrast, TCDD affects neither arachidonic acid release nor the turnover of phosphatidylinositol or phosphatidylcholine at any of the times tested. Although we examined biochemical effects which have been suggested as part of the mechanism of TCDD and which are produced by other epidermal proliferative compounds in XB cells, no mediator of the TCDD-produced differentiation of XB/3T3 cultures was observed.     

Lymphocyte labile iron pool,plasma iron,transferrin saturation and ferritin levels in colon cancer patients

Patients with colorectal carcinoma showed statistically significant lower values of transferrin saturation, total iron binding capacity and serum iron level as compared with control group, while the level of ferritin and the size of labile iron pool in carcinoma patients were higher, although this difference was not statistically significant. Our observations are in favour of the hypothesis which suggests that changes in iron metabolism restrict iron availability for tumour cells and as consequence, slow their growth.     

Stable expression of Anthozoa fluorescent proteins in mammalian cells

BACKGROUND: Fluorescent proteins have become invaluable reporters in many areas of cellular and developmental biology. An enhanced version of the Aequorea victoria green fluorescent protein (AvEGFP) is the most widely used fluorescent protein. For a variety of reasons, it is useful to have alternative fluorescent proteins to AvEGFP. METHODS: The cDNA sequences for enhanced variants of the Anemonia cyan fluorescent protein (AmCyan1), as well as the Zoanthus green (ZsGreen1) and yellow (ZsYellow1) fluorescent proteins, were cloned downstream of a constitutive cytomegalovirus (CMV) promoter within a retroviral expression vector. NIH3T3, HEK293, SW620, and WM35 cells were transduced with recombinant retroviruses at a low multiplicity of infection (MOI) to bias for single-copy integration. Both unselected and stably selected cells transduced with the retroviral expression constructs were characterized. Expression of each fluorescent protein in cells was detected using flow cytometry and fluorescence microscopy with filter sets typically used for AvEGFP/fluorescein isothiocyanate (FITC) detection and was compared with the expression of AvEGFP. In addition, a fluorescence plate reader with several excitation and emission filter sets was used for detection. RESULTS: Expression of each protein was observable by fluorescence microscopy. Under given conditions of flow cytometry, the ZsGreen1 mean fluorescence was approximately 3-fold, 10-fold, and 50-fold greater than that of AvEGFP, ZsYellow1, and AmCyan1, respectively. AmCyan1, ZsGreen1, and AvEGFP were detected by a fluorescence plate reader. CONCLUSION: We determined that fluorescent proteins from Anthozoa species are detectable using a standard flow cytometer and fluorescence microscope. All of the mammalian cell lines tested expressed detectable levels of fluorescent proteins from stable integrated provirus. In cell lines where the AvEGFP protein is toxic or poorly expressed, these Anthozoa fluorescent proteins may serve as alternative fluorescent reporters.     

Analysis of yggX and gshA mutants provides insights into the labile iron pool in Salmonella enterica

Strains of Salmonella enterica lacking YggX and the cellular reductant glutathione exhibit defects similar to those resulting from iron deficiency and oxidative stress. Mutant strains are sensitive to hydrogen peroxide and superoxide, deregulate the expression of the Fur-regulated gene entB, and fail to grow on succinate medium. Suppression of some yggX gshA mutant phenotypes by the cell-permeable iron chelator deferoxamine allowed the conclusion that increased levels of cellular Fenton chemistry played a role in the growth defects. The data presented are consistent with a scenario in which glutathione acts as a physiological chelator of the labile iron pool and in which YggX acts upstream of the labile iron pool by preventing superoxide toxicity.     

Expression of iron-related proteins in feline and canine mammary gland reveals unexpected accumulation of iron

Dysregulation of cellular iron homeostasis in human breast cancer is reflected by the altered expression of regulatory proteins. The expressions of iron-related proteins in the mammary glands of cats and dogs have not been assessed. We evaluated the expressions of ferritin, ferroportin, hepcidin and transferrin receptor 1 in benign and malignant mammary gland lesions in cats and dogs. Iron deposition was detected using Perls’ Prussian blue staining. We found no major differences in the expression of iron-related proteins between benign and malignant mammary gland lesions in either cats or dogs; however, these species exhibited accumulation of iron in benign lesions. Our findings provide an explanation for the absence of higher iron requirements by tumor cells in these animals. Further investigation of local iron homeostasis in cats and dogs and differences in their physiology compared to human breast cancer is required.     

Increased IRP1 and IRP2 RNA binding activity accompanies a reduction of the labile iron pool in HFE-expressing cells.

Iron regulatory proteins (IRPs), the cytosolic proteins involved in the maintenance of cellular iron homeostasis, bind to stem loop structures found in the mRNA of key proteins involved iron uptake, storage, and metabolism and regulate the expression of these proteins in response to changes in cellular iron needs. We have shown previously that HFE-expressing fWTHFE/tTA HeLa cells have slightly increased transferrin receptor levels and dramatically reduced ferritin levels when compared to the same clonal cell line without HFE (Gross et al., 1998, J Biol Chem 273:22068-22074). While HFE does not alter transferrin receptor trafficking or non-transferrin mediated iron uptake, it does specifically reduce (55)Fe uptake from transferrin (Roy et al., 1999, J Biol Chem 274:9022-9028). In this report, we show that IRP RNA binding activity is increased by up to 5-fold in HFE-expressing cells through the activation of both IRP isoforms. Calcein measurements show a 45% decrease in the intracellular labile iron pool in HFE-expressing cells, which is in keeping with the IRP activation. These results all point to the direct effect of the interaction of HFE with transferrin receptor in lowering the intracellular labile iron pool and establishing a new set point for iron regulation within the cell.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) enhances antibody production and protein kinase activity in murine B cells

Treatment of murine spleen cells with 30 nM TCDD resulted in an approximately 3 fold increase in unstimulated antibody production after 3 days in culture. This response was not accompanied by increased cellular proliferation and may represent an effect of TCDD on B cell activation or differentiation. Since PMA is capable of activating B cells, presumably via PKC, we have compared the effects of PMA and TCDD on protein kinase activation and phosphorylation of endogenous proteins in a highly purified preparation of B cells. In contrast to a reduction of cytosolic PKC activity, the expected effect of PMA, TCDD caused an increase in basal kinase activity with no effect on PKC activity. Addition of either PMA or TCDD resulted in enhanced phosphorylation of a similar profile of proteins, including proteins of Mr 12.2, 14.6, 29.2, 52.3 and 62.7 KDa. Addition of TCDD also resulted in the increased phosphorylation of a protein of Mr 45.2, which was unaffected by PMA. Combined treatment with PMA and TCDD resulted in additive responses. The additive effects of PMA and TCDD suggest an interaction at the level of protein phosphorylation which is mediated by different kinases. Therefore, TCDD may be stimulating B cells via an early effect on an unidentified protein kinase.