Addition Of Deferoxamine Research Articles

Agar and Chitosan Hydrogels' Design for Metal-Uptaking Treatments.

In the field of cultural heritage, the use of natural gels is rising for the application of active agents. Here, two natural polymers are assessed: agar, a pioneer hydrogel for conservation treatments, and chitosan, a rather novel and metal-binding gel. For chitosan, a state-of-the-art based formulation (CS-ItA-LCys) is evaluated as it was reported for silver-complexing properties. It is evaluated whether these polymers can withstand the addition of the chelating compound deferoxamine, which is a bacterial siderophore. This allows for the obtainment of completely bio-sourced gel systems. A Fourier-transformed (FT) infrared spectroscopy characterization is performed, completed with rheological measurements and Cryo-Scanning Electron Microscopy (cryo-SEM) to investigate the physico-chemical properties of the gels, as well as their interaction with deferoxamine. Both polymers are also tested for their inherent complexing ability on silver ions using FT-Raman spectroscopy. A multi-analytical comparison shows different microstructures, in particular, the presence of a thick membrane for chitosan and different mechanical behaviors, with agar being more brittle. Neither hydrogel seems affected by the addition of deferoxamine; this is shown by similar rheological behavior and molecular structures in the presence or absence of the chelator. The intrinsic abilities of the chitosan formulation to make silver complex are demonstrated with the observation of two peaks characteristic of Ag-S and Ag-O bonds. Agar and chitosan are both proven to be reliable gels to act as carriers for bio-based active agents. This paper confirms the potential asset of the chitosan formulation CS-ItA-LCys as a promising gel for the complexation of soluble silver.

Microcystin-LR prenatal exposure drives preeclampsia-like changes in mice by inhibiting the expression of TGF-β and VEGFA

Microcystin-leucine-arginine (MC-LR) is widespread in the water and food, which has suspected to be associated with adverse pregnancy outcomes. In the present study, we aim to assess the interaction between MC-LR exposure and preeclampsia development and elucidate the molecular events involved. After exposure to MC-LR during pregnancy, the mice developed hypertension and proteinuria, the typical symptoms of preeclampsia. This was associated with decreased invasiveness of placental trophoblast and vascular dysplasia caused by MC-LR through down-regulating VEGFA and TGF-β expression via AKT/m-TOR/HIF-1α pathway. In addition, this conclusion has been confirmed in a case-control study. Significantly, the addition of Deferoxamine (DFM), a phosphorylated serine-threonine protein kinases (p-AKT) specific agonist, can antagonize the inhibitory effect of MC-LR on the expression of related proteins, which further ameliorate the migration and invasion ability of HTR-8/Svneo cells. To sum up, our study revealed the pathologic mechanism by which MC-LR lead to preeclampsia and emphasized the importance of pregnancy management.

Abstract 2430: The role of cellular iron and the homeostatic iron regulator (HFE) in high-grade brain tumor cell migration

Abstract High-grade brain tumors such as grade III astrocytoma and glioblastoma represent among the most difficult to manage malignancies facing oncological practice. Diffuse migration and invasion into adjacent healthy brain tissue yields complete surgical resection of these tumors unfeasible. As a result, despite receiving maximal surgical resection and an aggressive course of chemoradiation, the majority of high-grade brain tumor patients suffer from recurrent disease. Increased expression levels of the homeostatic iron regulator gene (HFE) in high-grade brain tumors have been correlated with poorer outcomes. HFE is known to influence cellular iron metabolism by inhibiting transferrin-mediated iron uptake yet little is known regarding how HFE or iron impact the migratory capabilities of high-grade brain tumor cells. In order to better understand how HFE expression and cellular iron metabolism influence cell migration in high grade brain tumors, we utilized brain tumor cell lines that had been genetically manipulated to express different levels of HFE. We observed that knocking down HFE in KR158 or LN229 glioma cell lines resulted in significantly decreased migratory capacity. Since HFE is known to inhibit transferrin mediated iron uptake, we studied how directly modulating the iron status of glioma cells impacted their ability to migrate. Treatment of a panel of glioma cell lines: LN229, T98G, U87, KR158, with iron in the form of hemin or ferric ammonium citrate resulted in significantly reduced migration. Furthermore, the iron-induced reduction in migration could be rescued by the addition of deferoxamine, an iron chelator. Cell viability in response to the iron treatments was assayed and found to not be significantly altered - suggesting that cellular iron status was influencing migratory capacity independent of cell viability. To gain mechanistic insights into HFE and iron-induced effects on cell migration, we analyzed the Chinese Glioma Genome Atlas (CGGA) for correlations between HFE and the Rho GTPases RHOA, RAC1, and CDC42 – genes which are known to play a crucial role in determining the migratory capacity of cancer cells. Interestingly, we found statistically significant correlations between the Rho GTPases RHOA, RAC1, CDC42 and HFE in both grade III astrocytoma and glioblastoma patient cohorts. Immunoblotting of iron treated glioma cell lines demonstrated that expression of RhoA and Cdc42 was reduced suggesting that alterations in Rho GTPase expression and signaling may play a role in iron-induced effects on cell migration. Our results demonstrate that targeting cancer cell iron metabolism as an addition to existing treatment regimens may be a promising avenue for further investigation. Citation Format: Ganesh Shenoy, Katie Troike, Kondaiah Palsa, Madison Kuhn, Quinn Wade, Becky Slagle-Webb, Amanda Snyder, Chachrit Khunsriraksakul, Justin D. Lathia, Dhimant Desai, Hong-Gang Wang, Elizabeth Proctor, James R. Connor. The role of cellular iron and the homeostatic iron regulator (HFE) in high-grade brain tumor cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2430.

Effect of iron overload on endothelial cell calcification and its mechanism.

BackgroundVascular calcification is related to many diseases. Iron has a certain relationship with endothelial cells and vascular calcification. The purpose of this study was to assess the effect of iron overload on endothelial cell calcification and related mechanisms through cell experiments.MethodsHuman umbilical vein endothelial cells were treated with different concentrations of FeSO4 (50, 100, 150, and 200 µM), and deferoxamine (DFO) and ferrostatin. Alkaline phosphatase activity, malondialdehyde (MDA) level, reactive oxygen species (ROS) level, and lipid superoxidation after FeSO4 treatment were assessed. Alizarin red staining was used to observe calcium deposition. Quantitative polymerase chain reaction (qPCR) and western blot were adopted to examine the expression of calcification markers, iron metabolism-related factors, apoptosis pathway-related factors and ferroptosis markers. The TUNEL method was employed to detect cell apoptosis.ResultsFeSO4 of 100 µM significantly promoted the occurrence of cell ferroptosis, increased the levels of MDA and ROS, and decreased the ratio of glutathione (GSH) or glutathione disulfide (GSSG) and the expression level of glutathione peroxidase (GPX4). The addition of DFO and ferrostatin significantly modified the effects of FeSO4. Calcium deposition was most obvious in the cells treated with 100 µM FeSO4. FeSO4 significantly upregulated Runt-related transcription factor 2 (RUNX2) and Bone morphogenetic protein 2 (BMP2), ferritin heavy chain (FTH) and ferritin light chain (FTL), and downregulated Matrix Gla Protein (MGP) and divalent metal transporter 1 (DMT1). The results also showed that FeSO4 induced cell apoptosis by TUNEL method. The elevated Bcl2-associated death protein (Bad) and Bcl2-associated X protein (Bax) and the reduction in Bcl-2, p-Bad, p-AKT, and t-AKT were found. DFO and ferrostatin significantly reduced the iron-induced calcification and apoptosis of endothelial cells. DFO significantly increased the expression level of Bcl-2, and reduced the expression level of Bad.ConclusionsIron overload contributes to the process of endothelial cell calcification by inducing apoptosis and ferroptosis. Iron chelators and ferroptosis inhibitors alleviate endothelial cell apoptosis, ferroptosis, and calcification induced by iron overload.

Probing Hexaminolevulinate Mediated PpIX Fluorescence in Cancer Cell Suspensions in the Presence of Chemical Adjuvants.

Exogenous administration of hexaminolevulinate (HAL) induces fluorescent protoporphyrin IX (PpIX) accumulation preferentially in cancer cells. However, the PpIX fluorescence intensities between noncancer and cancer cells are highly variable. The contrast between cancer and noncancer cells may be insufficient to reliably discriminate, especially at the single cell level in cancer diagnostics. This study examines the use of the chemical adjuvants dimethylsulphoxide (DMSO) or deferoxamine (DFO) to enhance the HAL induced PpIX accumulation in cancer cells. Our results showed that in some of the incubation conditions tested, the addition of DFO with HAL significantly increased PpIX 21 fluorescence of adherent monolayer cancer cells, but this was never the case for cells in suspension. Permeabilisation with DMSO did not increase PpIX fluorescence. Cell-to-cell interaction may well play an important role in the PpIX accumulation when suspended cells are treated in HAL and adjuvant chemicals.

A nanocellulose-based colorimetric assay kit for smartphone sensing of iron and iron-chelating deferoxamine drug in biofluids

The current work describes the development of a “nanopaper-based analytical device (NAD)”, through the embedding of curcumin in transparent bacterial cellulose (BC) nanopaper, as a colorimetric assay kit for monitoring of iron and deferoxamine (DFO) as iron-chelating drug in biological fluids such as serum blood, urine and saliva. The iron sensing strategy using the developed assay kit is based on the decrease of the absorbance/color intensity of curcumin-embedded in BC nanopaper (CEBC) in the presence of Fe(III), due to the formation of Fe(III)-curcumin complex. On the other hand, releasing of Fe(III) from Fe(III)-CEBC upon addition of DFO as an iron-chelating drug, due to the high affinity of this drug to Fe(III) in competition with curcumin, which leads to recovery of the decreased absorption/color intensity of Fe(III)-CEBC, is utilized for selective colorimetric monitoring of this drug. The absorption/color changes of the fabricated assay kit as output signal can be monitored by smartphone camera or by using a spectrophotometer. The results of our developed sensor agreed well with the results from a clinical reference method for determination of Fe(III) concentration in human serum blood samples, which revealed the clinical applicability of our developed assay kit. Taken together, regarding the advantageous features of the developed sensor as an easy-to-use, non-toxic, disposable, cost-effective and portable assay kit, along with those of smartphone-based sensing, it is anticipated that this sensing bioplatform, which we name lab-on-nanopaper, will find utility for sensitive, selective and easy diagnosis of iron-related diseases (iron deficiency and iron overload) and therapeutic drug monitoring (TDM) of iron-chelating drugs in clinical analysis as well.

Abstract: Implantable Deferoxamine Facilitates Non-Vascularized Grafting in Irradiated Bone

INTRODUCTION: The goal of this proposal is to therapeutically reverse the damaging effects of radiotherapy on bone formation and healing to enable non-vascularized grafting in irradiated bone. Utilizing a rodent model of mandibular bone grafting, we quantified metrics of diminished graft take and bone healing in response to radiation treatment. Subsequently, we utilized implantable deferoxamine (DFO)-an angiogenic stimulant, to reverse these radiation-induced detriments. We hypothesized that the addition of our proposed therapy, would evidence quantifiable degrees of remediation on the process of tissue regeneration, graft incorporation and bone healing. METHODS: Male Lewis rats received a human equivalent dose of radiotherapy (7Gy/d x 5d) to left hemi-mandibles. After recovery, a circular trephine burr (6mm) was utilized to create a critical size defect just posterior to the third molar, and a bone graft was harvested from the right hemi-mandible of the same animal and secured with a custom PLA resorbable plate. Three groups (n=8/group) of animals were investigated: Control, (irradiated) XRT and irradiated + implantable deferoxamine (DFO). Mandibles were imaged at 14, 40 and 60 days with in vivo µCT, and a 60-day healing period was allowed prior to further outcomes testing. Bony union was judged clinically by 3 blinded reviewers on a scale from 0 to 4, representing the approximate percentage of robust union formation along the circular graft-recipient site interface (e.g. 1=25%, 4=100%). Statistical comparisons were conducted with ANOVA (p<0.05). RESULTS: We observed a significant diminution of bone graft healing after radiotherapy. At 60 days, the bone volume fraction (BVF) of the XRT group decreased by 20% (p=0.001), and exhibited lower bony union scores when compared to control (p=0.005). With the addition of DFO, these findings were largely remediated. At 60 days, the BVF improved upon the XRT BVF by 12% (p=0.025), and was not different than control (p=0.282). In addition, the bony union scores of the implanted DFO group significantly improved from XRT levels (p=0.05), and were not different than control (p=0.200). CONCLUSION: Implantable DFO strongly remediates the effects of radiation on non-vascularized bone graft incorporation and healing as measured by micro-densitometry and bony union analysis. These observations are promising with regards to the potential utility of this therapy to enhance bone graft incorporation in the irradiated mandible for head and neck cancer survivors.

Neurotoxic effects of iron overload under high glucose concentration.

Iron overload can lead to cytotoxicity, and it is a risk factor for diabetic peripheral neuropathy. However, the underlying mechanism remains unclear. We conjectured that iron overload-induced neurotoxicity might be associated with oxidative stress and the NF-E2-related factor 2 (Nrf2)/ARE signaling pathway. As an in vitro cellular model of diabetic peripheral neuropathy, PC12 cells exposed to high glucose concentration were used in this study. PC12 cells were cultured with ferric ammonium citrate at different concentrations to create iron overload. PC12 cells cultured in ferric ammonium citrate under high glucose concentration had significantly low cell viability, a high rate of apoptosis, and elevated reactive oxygen species and malondialdehyde levels. These changes were dependent on ferric ammonium citrate concentration. Nrf2 mRNA and protein expression in the ferric ammonium citrate groups were inhibited markedly in a dose-dependent manner. All changes could be inhibited by addition of deferoxamine. These results indicate that iron overload aggravates oxidative stress injury in neural cells under high glucose concentration and that the Nrf2/ARE signaling pathway might play an important role in this process.

Deferoxamine Restores Callus Size, Mineralization, and Mechanical Strength in Fracture Healing after Radiotherapy

Therapeutic augmentation of fracture-site angiogenesis with deferoxamine has proven to increase vascularity, callus size, and mineralization in long-bone fracture models. The authors posit that the addition of deferoxamine would enhance pathologic fracture healing in the setting of radiotherapy in a model where nonunions are the most common outcome. Thirty-five Sprague-Dawley rats were divided into three groups. Fracture, irradiated fracture, and irradiated fracture plus deferoxamine. The irradiated fracture and irradiated fracture plus deferoxamine groups received a human equivalent dose of radiotherapy [7 Gy/day for 5 days, (35 Gy)] 2 weeks before mandibular osteotomy and external fixation. The irradiated fracture plus deferoxamine group received injections of deferoxamine into the fracture callus after surgery. After a 40-day healing period, mandibles were dissected, clinically assessed for bony union, imaged with micro-computed tomography, and tension tested to failure. Compared with irradiated fractures, metrics of callus size, mineralization, and strength in deferoxamine-treated mandibles were significantly increased. These metrics were restored to a level demonstrating no statistical difference from control fractures. In addition, the authors observed an increased rate of achieving bony unions in the irradiated fracture plus deferoxamine-treated group when compared with irradiated fracture (67 percent and 20 percent, respectively). The authors' data demonstrate nearly total restoration of callus size, mineralization, and biomechanical strength, and a threefold increase in the rate of union with the use of deferoxamine. The authors' results suggest that the administration of deferoxamine may have the potential for clinical translation as a new treatment paradigm for radiation-induced pathologic fractures.

Coordination of iron ions in the form of histidinyl dinitrosyl complexes does not prevent their genotoxicity

Formation of dinitrosyl iron complexes (DNICs) was observed in a wide spectrum of pathophysiological conditions associated with overproduction of NO. To gain insight into the possible genotoxic effects of DNIC, we examined the interaction of histidinyl dinitrosyl iron complexes (HIS-DNIC) with DNA by means of circular dichroism. Formation of DNIC was monitored by EPR and FT/IR spectroscopy. Vibrational bands for aquated HIS-DNIC are reported. Dichroism results indicate that HIS-DNIC changes the conformation of the DNA in a dose-dependent manner in 10mM phosphate buffer (pH 6). Increase of the buffer pH or ionic strength decreased the effect. Comparison of HIS-DNIC DNA interaction with the effect of hydrated Fe2+ ion revealed many similarities. The importance of iron ions in HIS-DNIC induced genotoxicity is confirmed by plasmid nicking assay. Treatment of pUC19 plasmid with 1μM HIS-DNIC did not affect the plasmid supercoiling. Higher concentrations of HIS-DNIC induced single strand breaks. The effect was completely abrogated by addition of deferoxamine, a specific strong iron chelator. Our data reveal that formation of HIS-DNIC does not prevent DNA from iron-induced damage and imply that there is no direct interrelationship between iron–NO coordination and their mutual toxicity modulation.

In Vitro GDF15 Expression During Thalassemic Erythroid Differentiation and Maturation

Abstract 5285 INTRODUCTION:The growth differentiation factor-15 (GDF-15), a member of the transforming growth factor-β superfamily, is thought to be related to ineffective or apoptotic erythropoiesis. GDF-15 levels were found to be significantly elevated in sera of patients with β thalassemia major (TM), however little is known about the GDF15 expression during thalassemic erythropoiesis. Non-transfusion dependent thalassemia intermedia (NTDT) represents the model of thalassemic erythropoiesis not affected by transfusions. AIM:To determine the GDF15 gene expression profile during normal and thalassemic erythroid differentiation in standard cultures and under different iron conditions, from CD34+ of normal and thalassemia intermedia subjects. METHODS:After informed consent, the CD34+ cells were obtained from peripheral blood of healthy volunteers and from patients with NTDT by positive selection using anti-CD34-tagged magnetic beads and cultured for 14 days with a medium containing stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin to induce erythroid differentiation. Each culture was split in 3 flasks: standard condition, with addition of deferoxamine (DFO 4 mM) as iron chelating agent and ferric ammonium citrate (FAC 100 mM) at day 0 of culture. The expression profiling of GDF15 gene was evaluated at baseline, day 7 and day 14 by real-time PCR (2^-dCt). GDF15 concentrations in culture supernatants were also evaluated by enzyme-linked immunosorbent assay using DuoSet Sandwich ELISA Kit (R&D Systems, Minneapolis, MN). RESULTS:GDF15 expression and secretion increased significantly during erythroid differentiation either in normal and in NTDT cultures. At day 14 in thalassemia intermedia cultures GDF15 expression as well as the concentrations in supernatant were higher (althoug not statistically significant) compared to control (Table 1). At day 14 in control cultures GDF15 expression is up-regulated by DFO and down-regulated by FAC addition. In NTDT GDF15 expression was influenced by iron addition in cultures, but was not increased by iron depletion (Table 2).Table 12^-dCtpg/mLDay 0Day 7Day 14Day 7Day 14control0,0 ± 0,00,03 ± 0,01*8,13 ± 2,36*188 ± 82,73°8463 ± 3816°NTDT0,2 ± 0,01,24 ± 0,15°13,72 ± 3,68°229 ± 125*10005 ± 2114**p<0.001°p<0.05Table 2Day 142^-dCtcontrolNTDTuntreated8,13 ± 2,3613,72 ± 3,68DFO14,14 ± 0,10,58 ± 0,58FAC2,91 ± 0,51,13 ± 0,36 CONCLUSIONS:GDF15 levels in erythroid cultures are related to the erythropoietic stage of differentiation. In NTDT cultures the GDF15 gene profile and protein levels in supernatants are higher than in normal cultures. GDF15 levels seem to be modulated by iron in normal cultures whereas in NTDT cultures they seem to be independent from iron availability. This in vitro study supports that signals different than iron, such as erythropoietic stress, could be the major factor regulating GDF15 expression. Disclosures:No relevant conflicts of interest to declare.

Novel Cytochrome P450 Bioactivation of a Terminal Phenyl Acetylene Group: Formation of a One-Carbon Loss Benzaldehyde and Other Oxidative Products in the Presence of N-Acetyl Cysteine or Glutathione

Compounds 1 (N1-(3-ethynylphenyl)-6-methyl-N5-(3-(6-(methylamino)pyrimidin-4-yl)pyridin-2-yl) isoquinoline-1,5-diamine) and 2 (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine; Erlotinib/Tarceva) are kinase inhibitors that contain a terminal phenyl acetylene moiety. When incubated in the presence of P450 and NADPH, the anticipated phenyl acetic acid metabolite was formed. When 10 mM of N-acetyl-l-cysteine was added to the incubation mixtures, the phenyl acetic acid product was reduced and at 25 mM or higher concentration of NAC, formation of the phenyl acetic acid was abolished. Instead, the phenyl acetylene moiety lost a carbon and formed a benzaldehyde product. Other oxidation products incorporating one or more equivalents of NAC were also observed. The identities of the metabolites were characterized by MS and NMR. Addition of deferoxamine or ascorbic acid diminished the formation of the NAC influenced products. Similar products were also observed when 1 or 2 were incubated in P450 reactions supplemented with GSH, in Fenton reactions supplemented with NAC or GSH, and in peroxidase reactions supplemented with NAC. We propose the thiols act as a pro-oxidant readily undergoing a one-electron oxidation to form thiyl radicals which in turn initiates the formation of other peroxy radicals that drive the reaction to the observed products. These in vitro findings suggest that one-electron oxidation of thiols may promote the cooxidation of xenobiotic substrates.

Enhanced neurogenesis from neural progenitor cells with G1/S‐phase cell cycle arrest is mediated by transforming growth factor β1

We have previously demonstrated that a G1/S-phase cell cycle blocker, deferoxamine (DFO), increased the number of new neurons from rat neurosphere cultures, which correlated with prolonged expression of cyclin-dependent kinase (cdk) inhibitor p27(kip1) [H. J. Kim et al. (2006)Brain Research, 1092, 1-15]. The present study focuses on neuronal differentiation mechanisms following treatment of neural stem/progenitor cells (NPCs) with a G1/S-phase cell cycle blocker. The addition of DFO (0.5 mm) or aphidicolin (Aph) (1.5 microm) to neurospheres for 8 h, followed by 3 days of differentiation, resulted in an increased number of neurons and neurite outgrowth. DFO induced enhanced expression of transforming growth factor (TGF)-beta1 and cdk5 at 24 h after differentiation, whereas Aph only increased TGF-beta1 expression. DFO-induced neurogenesis and neurite outgrowth were attenuated by administration of a cdk5 inhibitor, roscovitine, suggesting that the neurogenic mechanisms differ between DFO and Aph. TGF-beta1 (10 ng/mL) did not increase neurite outgrowth but rather the number of beta-tubulin III-positive cells, which was accompanied by enhanced p27(kip1) mRNA expression. In addition, TGF-beta receptor type II expression was observed in nestin-positive NPCs. Results indicated that DFO-induced TGF-beta1 signaling activated smad3 translocation from the cytoplasm to the nucleus. In contrast, TGF-beta1 signaling inhibition, via a TGF-beta receptor type I inhibitor (SB-505124), resulted in decreased DFO-induced neurogenesis, in conjunction with decreased p27(kip1) protein expression and smad3 translocation to the nucleus. These results suggest that cell cycle arrest during G1/S-phase induces TGF-beta1 expression. This, in turn, prompts enhanced neuronal differentiation via smad3 translocation to the nucleus and subsequent p27(kip1) activation in NPCs.

The role of deferoxamine in the prevention of gentamicin ototoxicity: a histological and audiological study in guinea pigs

Conclusion. The addition of deferoxamine to gentamicin seems to confer partial functional and histological protection to the cochlea. Objective. Aminoglycosides are known ototoxic agents. The toxicity occurs via an activation process involving the formation of an iron–gentamicin complex with free radical production. Iron chelation will supposedly limit this toxic effect. This study aimed to determine the possible cochleoprotective role of deferoxamine on the ototoxic effect of gentamicin. Materials and methods. Sixty healthy active guinea pigs, weighing 400–600 g, with an average age of 6 months were used. They were divided into three groups. Group 1 received intramuscular gentamicin 8 mg/kg/day, group 2 received gentamicin 8 mg/kg/day and deferoxamine 150 mg/kg twice daily for 19 days and group 3 served as a control. All animals had a baseline measurement of distortion product autoacoustic emissions. At the end of 33 days they were submitted to another measurement and then the animals were sacrificed and their cochleas were examined histologically by light and transmission electron microscopy.Results. In group 1 the mean amplitude post-injection ranged from 5.83 dB at 1001 Hz to 22.33 dB at 6348 Hz. In the deferoxamine + gentamicin group the mean amplitude post-injection ranged from 5.10 dB at 1001 Hz, to 24.45 dB at 6348 Hz. This was statistically significant. At 4004, 5042 and 6348 Hz group 2 showed less histological damage than group 1.

Iron regulates T-lymphocyte sensitivity to the IFN-γ/STAT1 signaling pathway in vitro and in vivo

AbstractThe refractoriness of T cells to the interferon-γ (IFN-γ)/signal transducer and activator of transcription 1 (STAT1) pathway, which shields them from the antiproliferative effect of IFN-γ, is attributed mainly to down-regulation of the IFN-γR2 signaling chain. However, the mechanisms responsible for this down-regulation are unclear. Here we show that iron uptake mediated by the transferrin receptor (TfR) delivers a signal that leads to IFN-γR2 internalization and thus plays an essential role in attenuating activation of the IFN-γ/STAT1 pathway in human T lymphocytes. The effect of iron on IFN-γR2 internalization was specific as it did not affect expression of the IFN-γR1 binding chain. Deferoxamine (DFO), an iron-chelating agent, up-regulated IFN-γR2 surface expression and reinstated IFN-γ/STAT1 activation in proliferating T lymphocytes. Resistance of malignant T cells to the antiproliferative effect of IFN-γ in vitro was abrogated by addition of DFO. Conversely, iron inhibited IFN-γ–induced apoptosis in malignant T cells in serum-free conditions. In combination but not individually, DFO and IFN-γ strongly inhibited growth of human malignant T cells in an in vivo severe combined immunodeficient (SCID) mouse model. These data provide valuable insights for novel therapeutic approaches aimed at reinstating the IFN-γ/STAT1 apoptotic signaling pathway in autoreactive or neoplastic T cells by means of iron chelation.

Deferoxamine Reduces Cold-Ischemic Renal Injury in a Syngeneic Kidney Transplant Model

In cell-culture models, addition of deferoxamine (DFO) to University of Wisconsin Solution (UW solution) reduces cold-storage injury. The efficacy of DFO was therefore tested in a kidney transplantation model employing inbred Wistar Furth rats. Donor left kidneys, cold stored for 18 h in UW solution with or without 0.125 mM or 0.625 mM DFO were transplanted to the recipients' left renal fosse. Deferoxamine dose-dependently and significantly increased glomerular filtration rate (GFR) and renal blood flow (RBF), and suppressed renal F2-isoprostanes (vasoactive lipid peroxidation products) and apoptotic and necrotic injury 3 days post-transplantation. In a second set of similar experiments, the remaining native kidneys of the recipient rats were removed on day 7 of transplantation. Transplanted kidneys' function assessed by serum creatinine was 75% higher in the cold-stored transplanted kidneys treated with DFO compared with untreated kidneys. Moreover, the DFO treatment was attended by a significant reduction in apoptotic and necrotic tubular injury. Thus, our consistent findings from two sets of studies in a transplant model suggest that a simple strategy of including DFO in the cold-storage solution reduces cold ischemia-associated renal transplant damage and improves renal function. Our findings have potentially important ramifications for cold preservation of kidneys, and possibly other organs, in clinical transplantation.

GTP gamma S increases Nav1.8 current in small-diameter dorsal root ganglia neurons.

Tetrodotoxin-resistant (TTX-R) sodium current in small-size dorsal root ganglia (DRG) neurons is upregulated by prostaglandin E(2) and serotonin through a protein kinase A (PKA)/protein kinase (PKC) pathway, suggesting G protein modulation of one or more TTX-R channels in these neurons. Recently, GTP(gammaS), a hydrolysis-resistant analogue of GTP, was shown to increase the persistent current produced by the TTX-R Na(v)1.9. In this study, we investigated the modulation of another TTX-R channel, Na(v)1.8, by GTP(gammaS) in small-diameter DRG neurons from rats using whole-cell voltage clamp recordings. Because it has been suggested that fluoride, often used in intracellular recording solutions, may bind to trace amounts of aluminum and activate G proteins, we recorded Na(v)1.8 currents with and without intracellular fluoride, and with the addition of deferoxamine, an aluminum chelator, to prevent fluoride-aluminum binding. Our results show that GTP(gammaS) (100 micro M) caused a significant increase in Na(v)1.8 current (67%) with a chloride-based intracellular solution. Although the inclusion of fluoride instead of chloride in the pipette solution increased the Na(v)1.8 current by 177%, GTP(gammaS) further increased Na(v)1.8 current by 67% under these conditions. While the effect of GTP(gammaS) was prevented by pretreatment with H7 (100 micro M), a non-selective PKA/PKC inhibitor, the fluoride-induced increase in Na(v)1.8 current was not sensitive to H7 (100 micro M), or to inclusion of deferoxamine (1 mM) in the intracellular solution. We conclude that G protein activation by GTP(gammaS) increases Na(v)1.8 current through a PKA/PKC mechanism and that addition of fluoride to the pipette solution further enhances the current, but is not a confounding variable in the study of Na(v)1.8 channel modulation by G proteins independent of a PKA/PKC pathway or binding to aluminum.

Performance characteristics of the Dimension RxL iron and total iron-binding capacity methods

Background: Measurements of serum iron and total iron-binding capacity (TIBC) can be used to aid the diagnosis of iron deficiency and iron overload states. A variety of different methods for these measurements are commercially available. Methods: Linearity, imprecision and hemoglobin interference of homogeneous iron and TIBC methods on a Dimension® RxL analyzer were assessed. Method comparison studies were performed with a Vitros® 950 analyzer. Results: The Dimension RxL iron method was linear from 40 to 1000 μg/dl. The coefficient of variation of the iron and TIBC methods were <4% and <2%, respectively, at iron concentrations of 68 and 228 μg/dl and TIBC concentrations of 206 and 384 μg/dl. Comparison of the Vitros 950 and Dimension RxL iron methods gave a slope of 0.97, an intercept of 6.0 and r=0.99. Corresponding results for the TIBC methods were 1.02, −6.6 and r=0.97, respectively. Simulation of acute iron overload in vitro did not produce the same effect on TIBC measurements as in vivo overload. Iron recovery by the Dimension RxL method was reduced 40–60% by the addition of deferoxamine at a concentration of 200 μmol/l. Conclusions: The Dimension RxL assay provides acceptable measurements of iron and TIBC in routine patient samples. Iron measurements are unreliable in the presence of deferoxamine.

Calcein as a fluorescent iron chemosensor for the determination of low molecular weight iron in biological fluids.

The fluorescence quenching of calcein (CA) is not iron specific and results in a negative calibration curve. In the present study, deferoxamine (DFO), a strong iron chelator, was used to regenerate the fluorescence quenched by iron. Therefore, the differences in fluorescence reading of the same sample with or without addition of DFO are positively and specifically proportional to the amounts of iron. We found that the same iron species but different anions (e.g. ferric sulfate or ferric citrate) differed in CA fluorescence quenching, so did the same anions but different iron (e.g. ferrous or ferric sulfates). Excessive amounts of citrate competed with CA for iron and citrate could be removed by barium precipitation. After optimizing the experimental conditions, the sensitivity of the fluorescent CA assay is 0.02 microM of iron, at least 10 times more sensitive than the colorimetric assays. Sera from 6 healthy subjects were tested for low molecular weight (LMW) chelator bound iron in the filtrates of 10 kDa nominal molecular weight limit (NMWL). The LMW iron was marginally detectable in the normal sera. However, increased levels of LMW iron were obtained at higher transferrin (Tf) saturation (1.64-2.54 microM range at 80% Tf saturation, 2.77-3.15 microM range at 100% Tf saturation and 3.09-3.39 microM range at 120% Tf saturation). The application of the assay was further demonstrated in the filtrates of human liver HepG2 and human lung epithelial A549 cells treated with iron or iron-containing dusts.

Deferoxamine toxicity in hepatoma and primary rat cortical brain cultures.

Deferoxamine is commonly used for treatment of iron intoxication. Because the usual dose is unable to chelate sufficient iron before severe injury occurs, "high-dose" deferoxamine treatment has been proposed. However, several authors have reported severe toxicity after deferoxamine therapy. Although the hemodynamic effects are well described, the cellular toxicity of deferoxamine is unknown. Accordingly, we investigated the cellular toxicity of deferoxamine using in vitro techniques in two cell lines. Brain cells were harvested from fetal rats and cultured for 14-21 days before deferoxamine exposure. Using similar techniques, rat hepatoma cells were grown until confluent. Deferoxamine was added to the cultures to achieve final concentrations of 200-800 microg/ml, corresponding to in vivo infusion rates of 15-60 mg/kg/h. Deferoxamine was removed after 3 or 6 days by changing the medium. Subtoxic FeCl3 (500 mg/dl) was concurrently added to identical cultures to determine if deferoxamine potentiated iron toxicity. Cell viability was measured by a colorimetric assay. The addition of deferoxamine (0.2, 0.4, 0.8 mg/ml) significantly decreased cell viability in both cell groups. The effect of deferoxamine on primary cortical brain cultures was similar for the three concentrations used, and was similar when examined either 72 h or 6 days later. In contrast, hepatoma cell cultures evidenced a dose- dependent cell loss that increased with the length of exposure. The addition ofsubtoxic amounts of FeCl3 (500 microg/dl) in the presence of deferoxamine was protective in all cultures, and abolished deferoxamine-induced cell loss. Interestingly, the addition of serum albumin significantly reduced the amount of iron present in cells, suggesting its potential use to treat iron toxicity. These results suggest that deferoxamine, in the absence of iron, is toxic to cortical brain and hepatoma cells in vitro.