Concentrations Of Desferrioxamine Research Articles

Rahnella aquatilis JZ-GX1 alleviates iron deficiency chlorosis in Cinnamomum camphora by secreting desferrioxamine and reshaping the soil fungal community.

Plant growth-promoting rhizobacteria are important for improving plant iron nutrition, but the interactions among inoculants, host plants and soil microorganisms have not been greatly explored. Rahnella aquatilis JZ-GX1 was applied to treat the increasingly serious iron deficiency chlorosis in Cinnamomum camphora, and the resulting improvement in chlorosis was determined by assessing the contents of chlorophyll, active iron, Fe2+ and antioxidant enzymes in leaves, the effects on the soil microbial community and the metabolism in the rhizosphere by high-throughput sequencing techniques and liquid chromatography–mass spectrometry (LC–MS). The results showed that inoculation with JZ-GX1 significantly increased the chlorophyll content of C. camphora, which promoted the redistribution of active iron in roots and leaves, increased the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX), and thus reduced membrane damage in iron-deficient C. camphora caused by reactive oxygen species. According to genome prediction and ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) analysis, the JZ-GX1 strain could secrete desferrioxamine (DFO), and the concentration of DFO in C. camphora rhizosphere was 21-fold higher than that in uninoculated soil. The exogenous application of DFO increased the SPAD and Fe2+ contents in leaves. In addition, the inoculant affected the fungal community structure and composition in the C. camphora rhizosphere soil and increased the abundances of specific taxa, such as Glomus, Mortierella, Trichoderma, and Penicillium. Therefore, R. aquatilis JZ-GX1 application promoted iron absorption in C. camphora trees by secreting DFO and alleviated iron deficiency chlorosis through interactions with the local fungal community.

Hypoxia increases expression of selected blood\u2013brain barrier transporters GLUT-1, P-gp, SLC7A5 and TFRC, while maintaining barrier integrity, in brain capillary endothelial monolayers

BackgroundBrain capillary endothelial cells (BCECs) experience hypoxic conditions during early brain development. The newly formed capillaries are tight and functional before astrocytes and pericytes join the capillaries and establish the neurovascular unit. Brain endothelial cell phenotype markers P-gp (ABCB1), LAT-1(SLC7A5), GLUT-1(SLC2A1), and TFR(TFRC) have all been described to be hypoxia sensitive. Therefore, we hypothesized that monolayers of BCECs, cultured under hypoxic conditions, would show an increase in LAT-1, GLUT-1 and TFR expression and display tight endothelial barriers.Methods and resultsPrimary bovine BCECs were cultured under normoxic and hypoxic conditions. Chronic hypoxia induced HIF-1α stabilization and translocation to the nucleus, as judged by immunocytochemistry and confocal laser scanning imaging. Endothelial cell morphology, claudin-5 and ZO-1 localization and barrier integrity were unaffected by hypoxia, indicating that the tight junctions in the BBB model were not compromised. SLC7A5, SLC2A1, and TFRC-mRNA levels were increased in hypoxic cultures, while ABCB1 remained unchanged as shown by real-time qPCR. P-gp, TfR and GLUT-1 were found to be significantly increased at protein levels. An increase in uptake of [3H]-glucose was demonstrated, while a non-significant increase in the efflux ratio of the P-gp substrate [3H]-digoxin was observed in hypoxic cells. No changes were observed in functional LAT-1 as judged by uptake studies of [3H]-leucine. Stabilization of HIF-1α under normoxic conditions with desferrioxamine (DFO) mimicked the effects of hypoxia on endothelial cells. Furthermore, low concentrations of DFO caused an increase in transendothelial electrical resistance (TEER), suggesting that a slight activation of the HIF-1α system may actually increase brain endothelial monolayer tightness. Moreover, exposure of confluent monolayers to hypoxia resulted in markedly increase in TEER after 24 and 48 h, which corresponded to a higher transcript level of CLDN5.ConclusionsOur findings collectively suggest that hypoxic conditions increase some BBB transporters' expression via HIF-1α stabilization, without compromising monolayer integrity. This may in part explain why brain capillaries show early maturation, in terms of barrier tightness and protein expression, during embryogenesis, and provides a novel methodological tool for optimal brain endothelial culture.

The iron chelator desferrioxamine synergizes with chemotherapy for cancer treatment

BackgroundCisplatin (CDDP) resistance remains a major obstacle for treatment of ovarian cancer. Iron contributes to the growth and reproduction of malignant cells, thus iron chalators can inhibit the growth of tumor cells by depleting the intracellular iron pool. The iron chelator, desferrioxamine (DFO), has performed anticancer in previous study. The aim of our study is to determine the correlation between iron-deprivation and tumor chemosensitivity in ovarian cancer. MethodsTo investigate the prognostic value of ferritin light (FTL), ferroportin (FPN), hepcidin (HAMP) and divalent metal-ion transporter-1 (DMT1) in ovarian cancer, the Kaplan–Meier analysis and the Gene Expression Profiling Interactive Analysis (GEPIA) were used. The ovarian cancer cell lines (SKOV-3 and OVCAR-3) were exposed to a gradient concentration of DFO (10, 20, 50, 100, 200 μM) and CDDP (1, 5, 10, 50,100 μM) for 24 h. The protein expression of FTL was tested. The expression of cancer stem cell (CSC) markers, including Sox2, Nanog and C-myc, were downregulated with treatment of DFO. Also, the mamosphere formation and the plation of CD44+/high/CD133+/high and Aldehyde dehydrogenase (ALDH)+/high SKOV-3 cells were reduced after treatment for 7d. Furthermore, we detected the expression of p53, BCL-2, BAX, and caspase-8. ResultsThe survival analysis revealed that high expression of FTL, DMT1, HAMP, showed poor overall survival (OS) in ovarian cancer patients. Our combined data found that DFO could effectively inhibit CSCs, improve the resistance to chemotherapy, and significantly enhanced the efficacy of CDDP therapy in vitro in promoting apoptosis. Besides, targeting molecular targets, including BAX, BCL-2, p53 and caspase-8 could serve as the clinical biomarkers to evaluate the effects of ovarian cancer. It is reasonable to believe that DFO adjuvant therapy in combination with CDDP chemotherapy can promote the improvement of treatment response in ovarian cancer patients. ConclusionOur research suggests the experimental evidence for DFO and CDDP as a new effective combination therapy to enhance the efficacy of chemical therapy in ovarian cancer.

Effect of hydroxamate and catecholate siderophores on iron availability in the diatom Skeletonema costatum: Implications of siderophore degradation by associated bacteria

The bioavailability of iron (Fe) across marine ecosystems, mainly determined by Fe speciation and species-specific requirements of phytoplankton, remains largely unresolved. Siderophores are relevant within the pool of organic ligands that control organic Fe speciation. The effect on growth and physiology of the diatom Skeletonema costatum following addition over time of the uncomplexed siderophores (apo-form) desferrioxamine B and enterobactin were studied in the laboratory. The diatom was grown in batch culture in concentration gradients up to 50 and 10,000 nM for enterobactin and desferrioxamine B respectively. The potential effect of siderophore degradation was analyzed by electrospray ionization mass spectroscopy (HPLC-ESI-MS). Growth of S. costatum was negatively correlated to desferrioxamine concentration. In treatments where >500 nM was added, growth was negligible until day 9 after which significant growth started. Fe uptake at day 9 was highest at 10,000 nM, while the Fe quota was the lowest. The addition of enterobactin had a negative effect on the abundance, the in-vivo fluorescence and the Fe quota in S. costatum only at the highest concentration of 50 nM, while the in-vivo fluorescence was enhanced at the lowest concentration. The bacterial abundance over time was also negatively correlated to the concentration for both siderophores, but at day 9 the bacterial uptake showed an increase proportional to the siderophore concentration. HPLC-ESI-MS analysis revealed the presence of tentative metabolites of desferrioxamine in 500 and 10,000 nM indicating changes in concentration of the apo-siderophore. In the presence of cathecolate and hydroxamate siderophores, S. costatum exhibited the capacity for different Fe uptake strategies. The late growth exhibited and the high Fe uptake after prolonged Fe-limited growth, suggests that Fe reduction at cell's membrane may be facilitated by possible degradation of desferrioxamine by the associated bacteria. The results emphasize the need for studying Fe bioavailability of algae together with the interacting bacterial community.

Bioactive Injectable Hydrogels Containing Desferrioxamine and Bioglass for Diabetic Wound Healing.

Diabetic wound is hard to heal mainly because of the difficulty in vascularization in the wound area. Accumulating results have shown that desferrioxamine (DFO) can promote secretion of hypoxia inducible factor-1 (HIF-1α), thereby upregulating the expression of angiogenic growth factors and facilitating revascularization. Our preliminary study has demonstrated that Si ions in bioglass (BG) can upregulate vascular endothelial growth factor (VEGF) expression, thus promoting revascularization. It is hypothesized that the combined use of BG and DFO may have a synergistic effect in promoting VEGF expression and revascularization. To prove this, we first determined DFO concentration range that had no apparent cytotoxicity on human umbilical vein endothelial cells (HUVECs). Then, the optimal concentration of DFO promoting tube formation of HUVECs was determined by cell migration and tube formation assays. In addition, we demonstrated that combination use of BG and DFO improved the migration and tube formation of HUVECs as compared with the use of either BG or DFO alone as BG and DFO could synergistically upregulate VEGF expression. Furthermore, a sodium alginate hydrogel containing both BG and DFO was developed, and this hydrogel better facilitated diabetic skin wound healing than the use of either BG or DFO alone as BG and DFO in the hydrogels worked synergistically in promoting HIF-1α and VEGF expression and subsequently vascularization in the wound sites. Therefore, in this study, the synergistic effect in promoting revascularization between BG and DFO was first demonstrated and an injectable hydrogel simultaneously containing BG and DFO was developed for enhancing repair of diabetic chronic skin defects by taking advantages of the synergistic effects of BG and DFO in promoting revascularization. The study opens up a new prospect for the development of skin repair-promoting biomaterials.

Does Iron Modulate Cytokine Production by Desferrioxamine Treated Mononuclear Cells?

Iron is an essential element for maintaining the normal function of cellular and multicellular organisms and it is closely associated with inflammation and immunity. Macrophages and lymphocytes play an important role in its homeostasis. Hereby we have evaluated cytokine production by iron depleted and iron supplemented human peripheral blood mononuclear cells (PBMC) from healthy adult donors. Non-stimulated or stimulated PBMC (lipopolysaccharide-LPS or phorbol-myristate acetate-PMA and ionomycin) were incubated for 24 h without or with desferrioxamine (DFO) and the quantity of TNFα, IL-1β, IL-6, IFNγ, IL-2, IL-10 and IL-1ra in the supernatants was evaluated using ELISA kits. In addition, cytokine levels were determined in supernatants of cultures to which DFO was added for 2 h followed by 24 h of incubation with iron. The secretion of IL-1β, IL-6 and IL-1ra by non-stimulated PBMC and that of IL-1β by LPS-stimulated cells was dose-dependently enhanced following incubation with increasing concentrations of DFO between 25 and 100 µM. On the other hand LPS-induced production of IL-10 and PMA/ionomycin stimulated IL-2 and IFNγ secretion was dose dependently reduced. The inhibited IFNγ, IL-2 and IL-10 secretion by stimulated PBMC caused by DFO was reversed after incubation with iron at concentrations similar to those in human serum. However, the inhibition of LPS-induced IL-1ra or IL-1β production induced by DFO was not affected by iron supplementation. The results of the study underline the importance of intra and/or extracellular iron concentration and DFO for the release of inflammatory cytokines by human peripheral blood mononuclear cells.

A Potential Novel Application of Eltrombopag: A Combination Agent to Enhance Iron Chelation Therapy

INTRODUCTIONEltrombopag (ELT) is an orally available, non-peptide, small-molecule thrombopoietin receptor (TPO-R) agonist approved for the treatment of chronic immune thrombocytopenic purpura (ITP). Additionally ELT appears to bind intracellular iron (Roth et al, 2012, Blood) and our group has previously demonstrated its ability to progressively mobilize iron from cardiomyocytes in vitro. (Vlachodimitropoulou et al, Blood 2014, Volume 124, 21). The ELT concentrations at which iron was mobilized were substantially less (1µM) than with the clinically available iron chelators Desferrioxamine (DFO), Deferiprone (DFP) and Deferasirox (DFX), where 30µM iron binding equivalents (ibe) were required to achieve similar effects (Vlachodimitropoulou et al, 2014. Blood, Volume 124, 21). Importantly , the 1µM effective concentration of ELT for mobilizing cellular iron is nearly twenty-fold less than peak plasma concentrations reported clinically, even with low doses (30mg) of ELT (Gabianski, Journal of Clinical Pharmacology, 2011;51:842-856). At this low dose, increments in platelet counts do not typically exceed 1.2 x the baseline values in healthy volunteers with repeat dosing (Jenkins et al 2007, Blood, 109; 11 ). Hence it is predicted that effective chelating doses of ELT could be given without promoting unacceptable thombocytosis. In principle, still lower concentrations could be used for iron chelation if combined with another iron chelator. Here we explore and compare the concentrations at which effective cellular chelation is achieved with ELT alone or in combination with another chelator.METHODSAs cardiomyocytes are a target tissue for transfusional iron overload and provide a particular therapeutic challenge once iron has accumulated in them, the cardiomyocyte cell line H9C2, derived from embryonic rat ventricle, was chosen for investigation. As hepatocytes represent the cell type with the largest quantity of iron deposition, a human hepatocarcinoma HuH7 cell line was also evaluated. Cellular iron loading and iron mobilization were measured as a decrease in cellular iron content using the ferrozine assay (Vlachodimitropoulou et al 2015, British Journal of Haematology). The cells loaded with iron using 10% FBS containing media and then exposed to iron chelators/ELT. Cells were then lysed and intracellular iron concentration determined via the ferrozine assay, normalized against protein content. Acridine Orange/Propidium Iodide staining was used to ensure viability was consistently >98% during experiments, and to assess the toxicity of ELT on the cardiomyocyte and hepatocyte cell lines.RESULTSMonotherapy with 1µM ELT removed 42% of total cardiomyocyte iron following 8 hours of treatment. This was notably more efficient than in hepatocytes, where only 7% of cellular iron was removed with 1µM ELT monotherapy (Table 1). In Table 1 we can see the difference in iron removal between ELT monotherapy and combination with chelators after 8 hours. The effect in combination with all chelators was substantial. Viability was unaffected by combinations of 1µM ELT with other chelators. The hydrophilic hydroxypridinone iron chelator CP40, which has no iron mobilizing effects when used alone, enhanced iron mobilization by ELT, indicating that ELT can shuttle iron from cells onto a second chelator.CONCLUSIONRemarkably low concentrations of ELT monotherapy mobilize cellular iron from cardiomyocytes compared with conventional iron chelators. Furthermore, when used at as little as 1μΜ, in combination with standard therapeutic concentrations of DFO, DFP and DFX, the percentage of iron mobilized from cardiomyocytes more than doubled. Experiments with CP40 indicate that ELT acts as a shuttle molecule for chelated iron onto a second 'sink chelator' and that this is the likely mechanism for the enhanced iron mobilization with other iron chelators. While the action of ELT on the TPO-R is highly species-specific and occurs only in humans and primates, we found effective iron mobilization from both rat cardiomyocytes and human hepatocyte cell lines. This is consistent with an iron chelating mechanismdistinct from the TPO-R downstream signaling mechanism of ELT. The concentrations of ELT used to achieve iron mobilization in combination are clinically achievable and are unlikely to increase platelet counts in patients without thrombocytopaenia. DisclosuresPorter:Celgene: Consultancy; Shire: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding.

Influence and mechanism of mitochondrial permeability transition pore and cytochrome C in process of HL-60 cell apoptosis induced by desferrioxamine

Objective To explore the influence of mitochondrial permeability transition pore opening and cytochrome C(Cyt C) being discharged on the apoptotic mechanism of HL-60 cell induced by desferrioxamine(DFO), so as to provide scientific basis for the clinicians to adopt the strategy of iron deprivation to treat human leukemia. Methods HL-60 cells were co-cultivated with various concentration of DFO for 24-72 hours, then the apoptotic cells and the changes of mitochondrial membrane potential(ΔΨm) were examined by means of flow cytometry(FCM), and Cyt C in cytoplasm was detected by way of celluar immunohistochemistry. Results The cell apoptotic rate assumed rising ten-dency as the highest rate could be up to(44.10±6.31)%, and the effect was of time-and-dose dependence(P<0.01). FCM could detect the ΔΨm declining(P<0.05), and the Cyt C positive cell rate was higher than the controls, the differences were of statistical significance(P<0.05). Conclusions DFO can induce the HL-60 cells apoptosis, and the possible mechanism is that DFO make the mitochondrial permeability transition pore open and get Cyt C discharged from the mitochondria. Key words: Desferrioxamine; HL-60 cell; Apoptosis; Mitochondrion membrane potential; Cytochrome C

Development of a thermoresponsive chitosan gel combined with human mesenchymal stem cells and desferrioxamine as a multimodal pro-angiogenic therapeutic for the treatment of critical limb ischaemia

Critical limb ischaemia (CLI) is a debilitating ischaemic disease caused by vascular occlusion. Pro-angiogenic therapeutics have the potential to produce collateral vasculature, delaying or negating the need for amputation or invasive revascularisation. Thermoresponsive hydrogels can provide an in situ depot for the sustained release of drugs and provide protection and cohesion for encapsulated cells. Human mesenchymal stem cells (hMSCs) have demonstrated strong angiogenic potential in vitro and angiogenic efficacy in vivo. Desferrioxamine (DFO), a pharmacological activator of the pro-angiogenic hypoxia inducible factor-1α pathway, has shown pro-angiogenic efficacy in vivo. This study combined hMSCs and DFO with a thermoresponsive chitosan/β-glycerophosphate (β-GP) gel, to function as an injectable, multimodal, pro-angiogenic therapeutic for the treatment of CLI. This gel underwent a thermogelation beginning at 33°C, and provided a sustained, biologically active release of DFO over the space of seven days, whilst permitting the survival, proliferation and migration of encapsulated hMSCs. hMSCs encapsulated in gel containing a 100μM concentration of DFO displayed an upregulation in VEGF expression. The combination of hMSCs and DFO within the gel resulted in a synergistic enhancement in bioactivity, as measured by increased VEGF expression in gel-exposed human umbilical vein endothelial cells. This formulation displays significant potential as an injectable pro-angiogenic therapeutic for the treatment of CLI.

Preconditioning protects against oxidative injury involving hypoxia-inducible factor-1 and vascular endothelial growth factor in cultured astrocytes

Tolerance to brain injury involves hypoxia-inducible factor-1 (HIF-1) and its target genes as the key pathway mediating a cascade of events including cell survival, energetics, and angiogenesis. In this study, we established the treatment paradigms for an in vitro model of tolerance to oxidative injury in primary astrocytic cultures and further examined the roles for the HIF-1 signalling cascade. Isolated murine astrocytes were preconditioned with sub-toxic concentrations of HIF-1 inducers and subsequently exposed to a H 2O 2 insult, where changes in cell viability and protein expression were determined. Preconditioning with non-damaging concentrations of desferrioxamine (DFO) and ethyl-3,4-dihydroxybenzoate (EDHB) significantly improved cellular viability after H 2O 2 injury treatment. Time course studies revealed that DFO and EDHB treatments alone induced sequential activation of HIF-1 signal transduction where nuclear HIF-1α protein accumulation was detected as early as 2 h, followed by downstream upregulation of intracellular and released VEGF from 4 h and 8 h onwards, respectively. The protective effects of DFO and EDHB preconditioning against H 2O 2 injury were abolished by co-treatment with cycloheximide, an inhibitor of protein synthesis. Importantly, when the anti-HIF-1 compound, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) was used, the cytoprotection and VEGF accumulation produced by DFO and EDHB preconditioning were diminished. These results indicate the essential role of the HIF-1 pathway in our model of tolerance against oxidative injury in cultured astrocytes, and suggest roles for astrocytic HIF-1 expression and VEGF release which may influence the function of surrounding cells and vasculature during oxidative stress-related brain diseases.

Lactoferrin and desferrioxamine are ineffective in the treatment ofHelicobacter pyloriinfection and may enhanceH. pylorigrowth and gastric inflammation in mice

To evaluate the efficacy of bovine lactoferrin (BLf), recombinant human lactoferrin (rHLf) and desferrioxamine against Helicobacter pylori in vitro and in mice and also to determine whether BLf or rHLf alter gastric inflammation. In vitro: Broth dilution susceptibility tests were performed using different concentrations of desferrioxamine, BLf and rHLf. Murine trials: In the prevention trial, C57BL/6 female mice were treated with BLf or rHLF, and then infected with the SS1 strain of H. pylori. In the treatment trial, mice were gavaged with either BLf, rHLf or desferrioxamine. In addition, gastric myeloperoxidase activity (MPO) was measured to assess gastric inflammation. Desferoxamine was found to have a direct bactericidal effect, while BLf and rHLf only partially suppressed H. pylori growth in vitro. However, in both prevention and treatment trials all three forms of treatment failed to reduce H. pylori load in mice. Gastric MPO activity and H. pylori load were noted to be higher with lactoferrin treatments. Our study does not support the use of BLf or rHLF in the treatment of human H. pylori infection. Interestingly, H. pylori growth and gastric inflammation appear to be enhanced by lactoferrin treatment. The mouse model is ideal for testing novel H. pylori eradicating agents.

In vitro chelating, cytotoxicity, and blood compatibility of degradable poly(ethylene glycol)-based macromolecular iron chelators

Desferrioxamine (DFO) is used to treat an excess accumulation of iron in the body and is currently the most commonly used iron chelator for the treatment of ‘iron overload’ disorder. However, the disadvantages of DFO surround its high toxicity and very short plasma half-life. Here, the detailed in vitro evaluation of a novel class of high molecular weight iron chelators based on DFO and polyethylene glycol methacrylate is reported. Reversible addition fragment chain transfer (RAFT) copolymerization afforded polymer conjugates (P-DFO) with well-controlled molecular weight (27–127 kDa) and substitution of DFO (5–26 units per chain) along the copolymer. Human umbilical vein endothelial cell (HUVEC) based cell viability assays showed that the cytotoxicity of P-DFO decreased more than 100-fold at identical concentrations of DFO. The hemocompatibilities of various P-DFO samples were determined by measuring prothrombin time (PT), activated partial thromboplastin time (APTT), thrombelastograph parameters (TEG), complement activation, platelet activation, and red blood cell aggregation. Furthermore, the iron binding properties and chelating efficiency of P-DFO were compared to DFO by measuring the spectral properties upon binding to iron(III), while the prevention of iron(III) mediated oxidation of hemoglobin was also determined. Degradation of the P-DFO conjugates via cleavable ester linkages between the polymer backbone and the PEG side chains was evaluated using gel permeation chromatography (GPC) and NMR. Since the chelating ability of DFO remains intact after conjugation to the copolymer backbone, these macromolecular, blood compatible and degradable conjugates are promising candidates as long circulating, non-toxic iron chelators.

Direct Comparison of δ-Aminolevulinic Acid and Methyl-Aminolevulinate-Derived Protoporphyrin IX Accumulations Potentiated by Desferrioxamine or the Novel Hydroxypyridinone Iron Chelator CP94 in Cultured Human Cells

Aminolevulinic acid photodynamic therapy (ALA-PDT) is a cancer therapy that combines the selective accumulation of a photosensitizer in tumor tissue with visible light (and tissue oxygen) to produce reactive oxygen species. This results in cellular damage and ablation of tumor tissue. The use of iron chelators in combination with ALA has the potential to increase the accumulation of the photosensitizer protoporphyrin IX (PpIX) by reducing its bioconversion to heme. This study compares directly for the first time the effects of the novel hydroxypyridinone iron chelating agent CP94 and the more clinically established iron chelator desferrioxamine (DFO) on the enhancement of ALA and methyl-aminolevulinate (MAL)-induced PpIX accumulations in cultured human cells. Cultured human cells were incubated with a combination of ALA, MAL, CP94 and DFO concentrations; the resulting PpIX accumulations being quantified fluorometrically. The use of iron chelators in combination with ALA or MAL was shown to significantly increase the amount of PpIX accumulating in the fetal lung fibroblasts and epidermal carcinoma cells; while minimal enhancement was observed in the normal skin cells investigated (fibroblasts and keratinocytes). Where enhancement was observed CP94 was shown to be significantly superior to DFO in the enhancement of PpIX accumulation.

Down-regulation of dopamine transporter by iron chelationin vitrois mediated by altered trafficking, not synthesis

Neurological development and functioning of dopamine (DA) neurotransmission is adversely affected by iron deficiency in early life. Iron-deficient rats demonstrate significant elevations in extracellular DA and a reduction in dopamine transporter (DAT) densities in the caudate putamen and nucleus accumbens. To explore possible mechanisms by which cellular iron concentrations control DAT functioning, endogenous DAT-expressing PC12 cells were used to determine the effect of iron chelation on DAT protein and mRNA expression patterns. In addition, we used human DAT (hDAT)-transfected Neuro2a (N2A) cells to examine DAT degradation and trafficking patterns. A 50 microM treatment for 24 h with the iron chelator, desferrioxamine (DFO), significantly decreased dopamine uptake in a dose-dependent manner, with no apparent change in K(m), in both PC12 and N2A cells. Reduced DA uptake was accompanied by concentration- and time-dependent reductions in total DAT protein levels in both cell lines. Exposure to increasing concentrations of DFO did not significantly alter DAT mRNA in either PC12 or N2A cells. However, DAT degradation rates increased three-fivefold in both cell types exposed to 50 microM DFO for 24 h. Biotinylation studies in N2A cells indicate a more dramatic loss of DAT in the membrane fraction, while OptiPrep fractionation experiments revealed an increase in lysosomal DAT with iron chelation. Inhibition of protein kinase C activation with staurosporin prevented the effect of iron chelation on DAT function, suggesting that in vitro iron chelation affects DAT primarily through the effects on trafficking rather than on synthesis.

Outer‐membrane siderophore receptors of heterotrophic oceanic bacteria

Pathogenic gram‐negative bacteria use specific receptors to transport ferric siderophore complexes across their outer membrane during iron (Fe)‐limited growth. Receptors such as these have not yet been characterized in oceanic heterotrophic bacteria. We examined four species of γ‐proteobacteria for the presence of Fe‐siderophore receptors with the use of a nondenaturing polyacrylamide gel electrophoresis binding assay and the siderophore ferrioxamine B (FB) labeled with 55Fe. Small‐subunit rRNA sequence analysis assigned these bacteria to the genera Pseudoalteromonas and Alteromonas. Two oceanic species, Pseudoalteromonas haloplanktis (Neptune) and Alteromonas macleodii (Jul88), which were shown previously to transport and assimilate Fe bound to FB during growth, synthesized an outer‐membrane FB receptor under Fe‐limiting conditions. Only low concentrations of the receptors were detected in these bacteria when they were grown with high concentrations of Fe. The FB receptor of P. haloplanktis (Neptune) had an apparent molecular mass of 79 kDa and an externally oriented binding site. The molecular mass of the receptor of A. macleodii (Jul88) was 100 kDa. No FB receptors were detected by our methods in two coastal species, Pseudoalteromonas rubra (LMG1) and Pseudoalteromonas piscicida (PWF3). P. haloplanktis (Neptune) and A. macleodii (Jul88) also bound 55Fe‐ferrichrome, a trihydroxamate siderophore like FB. Binding assays conducted with 115 nmol L−1 55Fe‐FB in the presence of increasing concentrations of desferrioxamine B showed a progressive decrease in the amount of 55Fe‐FB bound by the receptor protein, suggesting strong affinity of the receptor for the Fe‐free siderophore. Our results provide the first demonstration of Fe‐siderophore receptors in oceanic heterotrophic bacteria.

β-Carotene 15,15′-Dioxygenase activity in human tissues and cells: evidence of an iron dependency

The two objectives of this study were to investigate β-carotene 15,15′-dioxygenase activity in human tissues and to determine the effect of desferrioxamine on the dioxygenase activity. Two human in vitro models were used: the TC7 clone of the intestinal cell line Caco-2 and small intestinal mucosa preparations. β-Carotene 15,15′-dioxygenase activity in the small intestinal mucosa was (mean ± SD) 97.4 ± 39.8 pmol/h.mg protein for five adults (44–89 y) and 20 pmol/h.mg for an infant (17 months). No activity was detected in adult stomach tissue. We report for the first time the dioxygenase activity in human liver: 62 pmol/h.mg for a normal adult liver and 7 pmol/h.mg for a liver exhibiting gross pathology. The maximum capacity of β-carotene cleavage in an adult was estimated to be 12 mg/day (one fifth by small intestine and four fifths by liver), assuming an optimal β-carotene/retinal cleavage ratio of 1:2. The dioxygenase activity was decreased up to 80% with increasing desferrioxamine concentrations in the two in vitro models. Desferrioxamine was characterized as a noncompetitive inhibitor. In TC7 cells, the inhibitory effect of desferrioxamine was reversed by iron addition, suggesting that this effect was related to the ability of desferrioxamine to chelate iron, purported to be an obligate cofactor of the enzyme. In conclusion, these data report the presence of β-carotene 15,15′-dioxygenase activity in human small intestine and liver and demonstrate that desferrioxamine efficiently inhibits intestinal β-carotene cleavage in human tissues and cells.

Hemoproteins affect H 2O 2 removal from rat tissues

The capacity of rat liver homogenates and mitochondria to remove H 2O 2 was determined by comparing their ability to slow fluorescence generated by a H 2O 2 ‘detector’ with that of desferrioxamine solutions. H 2O 2 was produced by glucose oxidase-catalysed glucose oxidation. The capacity to remove H 2O 2 was expressed as equivalent concentration of desferrioxamine. The method showed changes in the capacity of H 2O 2 removal after treatment with ter-butylhydroperoxide or glutathione. The H 2O 2 removal capacity of homogenates and mitochondria from rat liver, heart, and skeletal muscle was compared with their overall antioxidant capacity. For homogenates, the order of both antioxidant and H 2O 2 removal capacities was liver>heart>muscle. For mitochondria, the order of the antioxidant capacities mirrored that of the homogenates, while the order of the H 2O 2 removal capacities was heart>muscle>liver. Because H 2O 2 removal is not only due to H 2O 2-metabolyzing enzymes, but also to hemoproteins that convert H 2O 2 into more reactive radicals via Fenton reaction, the higher concentration of cytochromes in mitochondria of cardiac and skeletal muscles can explain the above discrepancy. A higher H 2O 2 removal capacity was found to be associated with a higher rate of H 2O 2 release by mitochondria, indicating that the order of H 2O 2 release rate mirrors that of H 2O 2 production rate. We suggest that the different capacities of the mitochondria from the three tissues to produce reactive oxygen species are due to differences in the concentration of respiratory mitochondrial chain components in the reduced form.

Inhibitors of poly (ADP-ribose) synthetase protect rat proximal tubular cells against oxidant stress

The generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of renal ischemia-reperfusion injury. ROS produce DNA strand breaks that lead to the activation of the DNA-repair enzyme poly (ADP-ribose) synthetase (PARS). Excessive PARS activation results in the depletion of its substrate, nicotinamide adenine dinucleotide (NAD) and subsequently of adenosine 5'-triphosphate (ATP), leading to cellular dysfunction and eventual cell death. The aim of this study was to investigate the effect of various PARS inhibitors on the cellular injury and death of rat renal proximal tubular (PT) cells exposed to hydrogen peroxide (H2O2). Rat PT cell cultures were incubated with H2O2 (1 mM) either in the presence or absence of the PARS inhibitors 3-aminobenzamide (3-AB, 3 mM), 1,5-dihydroxyisoquinoline (0.3 mM) or nicotinamide (Nic, 3 mM), or increasing concentrations of desferrioxamine (0.03 to 3 mM) or catalase (0.03 to 3 U/ml). Cellular injury and death were determined using the MTT and lactate dehydrogenase (LDH) assays, respectively. H2O2-mediated PARS activation in rat PT cells and the effects of PARS inhibitors on PARS activity were determined by measurement of the incorporation of [3H]NAD into nuclear proteins. Incubation of rat PT cells with H2O2 significantly inhibited mitochondrial respiration and increased LDH release, respectively. Both desferrioxamine and catalase reduced H2O2-mediated cellular injury and death. All three PARS inhibitors significantly attenuated the H2O2-mediated decrease in mitochondrial respiration and the increase in LDH release. Incubation with H2O2 produced a significant increase in PARS activity that was significantly reduced by all PARS inhibitors. 3-Aminobenzoic acid (3 mM) and nicotinic acid (3 mM), structural analogs of 3-AB and Nic, respectively, which did not inhibit PARS activity, did not reduce the H2O2-mediated injury and necrosis in cultures of rat PT cells. We propose that PARS activation contributes to ROS-mediated injury of rat PT cells and, therefore, to the cellular injury and cell death associated with conditions of oxidant stress in the kidney.

Regulation of Erythropoietin Gene Expression Depends on Two Different Oxygen-Sensing Mechanisms

Erythropoietin (Epo), a glycoprotein hormone produced principally in the fetal kidney and in the adult liver in response to hypoxia, is the prime regulator of growth and differentiation in erythroid progenitor cells. The regulation of Epo gene expression is not fully understood, but two mechanisms have been proposed. One involves the participation of a heme protein capable of reversible oxygenation and the other depends on the intracellular concentration of reactive oxygen species (ROS), assumed to be a function of pO2. We have investigated the production of Epo in response to three stimuli, hypoxia, cobalt chloride, and the iron chelator desferrioxamine, in Hep3B cells. As expected, hypoxia caused a marked rise in Epo production. When the cells were exposed to the paired stimuli of hypoxia and cobalt no further increase was found. In contrast, chelation of iron under hypoxic conditions markedly enhanced Epo production, suggesting that the two stimuli act by separate pathways. The addition of carbon monoxide inhibited hypoxia-induced Epo production, independent of desferrioxamine concentration. Taken together these data support the concept that pO2 and ROS are sensed independently.

Inhibitors of poly (ADP-ribose) synthetase protect rat renal proximal tubular cells against oxidant stress.

Inhibitors of poly (ADP-ribose) synthetase protect rat proxiIschemia followed by reperfusion of the kidney inmal tubular cells against oxidant stress. volves a complex and interrelated sequence of events Background. The generation of reactive oxygen species (ROS) that result in the injury of renal cells and eventual cell has been implicated in the pathogenesis of renal ischemiadeath due to apoptosis and necrosis [1, 2]. Although reperfusion injury. ROS produce DNA strand breaks that lead reperfusion is essential for the survival of ischemic tissue, to the activation of the DNA-repair enzyme poly (ADP-ribose) synthetase (PARS). Excessive PARS activation results in the there is good evidence that reperfusion itself causes addidepletion of its substrate, nicotinamide adenine dinucleotide tional cell injury (reperfusion-injury) [3], which has been (NAD) and subsequently of adenosine 59-triphosphate (ATP), attributed to calcium overload, neutrophil infiltration leading to cellular dysfunction and eventual cell death. The and generation of reactive oxygen species (ROS) [1–3]. aim of this study was to investigate the effect of various PARS inhibitors on the cellular injury and death of rat renal proximal ROS–mediated cell injury and death has been implicated tubular (PT) cells exposed to hydrogen peroxide (H2O2). in the pathogenesis of renal ischemia-reperfusion injury Methods. Rat PT cell cultures were incubated with H2O2 and associated renal failure [1, 2]. Within the kidney, (1 mm) either in the presence or absence of the PARS inhibitors the situation is particularly complex, as ischemia itself 3-aminobenzamide (3-AB, 3 mm), 1,5-dihydroxyisoquinoline can cause early irreversible damage, which appears to (0.3 mm) or nicotinamide (Nic, 3 mm), or increasing concentrations of desferrioxamine (0.03 to 3 mm) or catalase (0.03 to 3 be mediated by ROS [2]. Furthermore, the proximal U/ml). Cellular injury and death were determined using the tubule (PT) appears to be particularly susceptible to MTT and lactate dehydrogenase (LDH) assays, respectively. reperfusion-injury [1, 2]. H2O2-mediated PARS activation in rat PT cells and the effects ROS produce cellular injury and necrosis via several of PARS inhibitors on PARS activity were determined by mechanisms including peroxidation of membrane lipids, measurement of the incorporation of [H]NAD into nuclear proteins. protein denaturation and DNA damage [3]. Evidence obResults. Incubation of rat PT cells with H2O2 significantly tained using cultured cells demonstrate that ROS produce inhibited mitochondrial respiration and increased LDH restrand breaks in DNA, which triggers energy-consuming lease, respectively. Both desferrioxamine and catalase reduced DNA repair mechanisms and activates the nuclear enH2O2-mediated cellular injury and death. All three PARS inhibitors significantly attenuated the H2O2-mediated decrease zyme poly (ADP-ribose) synthetase (PARS) [EC 2.4.2.30, in mitochondrial respiration and the increase in LDH release. also referred to as poly (ADP-ribose) polymerase Incubation with H2O2 produced a significant increase in PARS (PARP) or poly (ADP-ribose) transferase (pADPRT)] activity that was significantly reduced by all PARS inhibitors. [4]. PARS is an abundant, chromatin-bound enzyme con3-Aminobenzoic acid (3 mm) and nicotinic acid (3 mm), strucstitutively expressed in numerous cell types [5], which tural analogs of 3-AB and Nic, respectively, which did not inhibit PARS activity, did not reduce the H2O2-mediated injury when activated catalyzes the transfer of ADP-ribose and necrosis in cultures of rat PT cells. moieties from NAD to nuclear proteins including hisConclusion. We propose that PARS activation contributes tones and onto PARS itself (automodification) with the to ROS-mediated injury of rat PT cells and, therefore, to the concomitant formation of nicotinamide [6]. However, cellular injury and cell death associated with conditions of oxidant stress in the kidney. there is now good evidence that exposure of cells to oxidant stress in the form of superoxide anions, hydrogen peroxide (H2O2) and hydroxyl radicals causes strand