The protective role of transferrin in Müller glial cells after iron-induced toxicity.

PURPOSE: Transferrin (Tf) expression is enhanced by aging and inflammation in humans. We investigated the role of transferrin in glial protection. METHODS: We generated transgenic mice (Tg) carrying the complete human transferrin gene on a C57Bl/6J genetic background. We studied human (hTf) and mouse (mTf) transferrin localization in Tg and wild-type (WT) C57Bl/6J mice using immunochemistry with specific antibodies. Müller glial (MG) cells were cultured from explants and characterized using cellular retinaldehyde binding protein (CRALBP) and vimentin antibodies. They were further subcultured for study. We incubated cells with FeCl(3)-nitrilotriacetate to test for the iron-induced stress response; viability was determined by direct counting and measurement of lactate dehydrogenase (LDH) activity. Tf expression was determined by reverse transcriptase-quantitative PCR with human- or mouse-specific probes. hTf and mTf in the medium were assayed by ELISA or radioimmunoassay (RIA), respectively. RESULTS: mTf was mainly localized in retinal pigment epithelium and ganglion cell layers in retina sections of both mouse lines. hTf was abundant in MG cells. The distribution of mTf and hTf mRNA was consistent with these findings. mTf and hTf were secreted into the medium of MG cell primary cultures. Cells from Tg mice secreted hTf at a particularly high level. However, both WT and Tg cell cultures lose their ability to secrete Tf after a few passages. Tg MG cells secreting hTf were more resistant to iron-induced stress toxicity than those no longer secreted hTf. Similarly, exogenous human apo-Tf, but not human holo-Tf, conferred resistance to iron-induced stress on MG cells from WT mice. CONCLUSIONS: hTf localization in MG cells from Tg mice was reminiscent of that reported for aged human retina and age-related macular degeneration, both conditions associated with iron deposition. The role of hTf in protection against toxicity in Tg MG cells probably involves an adaptive mechanism developed in neural retina to control iron-induced stress.

Tumor necrosis factors alpha and beta protect neurons against amyloid beta-peptide toxicity: evidence for involvement of a kappa B-binding factor and attenuation of peroxide and Ca2+ accumulation.

In Alzheimer disease (AD) the amyloid beta-peptide (A beta) accumulates in plaques in the brain. A beta can be neurotoxic by a mechanism involving induction of reactive oxygen species (ROS) and elevation of intracellular free calcium levels ([Ca2+]i). In light of evidence for an inflammatory response in the brain in AD and reports of increased levels of tumor necrosis factor (TNF) in AD brain we tested the hypothesis that TNFs affect neuronal vulnerability to A beta. A beta-(25-35) and A beta-(1-40) induced neuronal degeneration in a concentration- and time-dependent manner. Pretreatment of cultures for 24 hr with TNF-beta or TNF-alpha resulted in significant attenuation of A beta-induced neuronal degeneration. Accumulation of peroxides induced in neurons by A beta was significantly attenuated in TNF-pretreated cultures, and TNFs protected neurons against iron toxicity, suggesting that TNFs induce antioxidant pathways. The [Ca2+]i response to glutamate (quantified by fura-2 imaging) was markedly potentiated in neurons exposed to A beta, and this action of A beta was suppressed in cultures pretreated with TNFs. Electrophoretic mobility-shift assays demonstrated an induction of a kappa beta-binding activity in hippocampal cells exposed to TNFs. Exposure of cultures to I kappa B (MAD3) antisense oligonucleotides, a manipulation designed to induce NF-kappa B, mimicked the protection by TNFs. These data suggest that TNFs protect hippocampal neurons against A beta toxicity by suppressing accumulation of ROS and Ca2+ and that kappa B-dependent transcription is sufficient to mediate these effects. A modulatory role for TNF in the neurodegenerative process in AD is proposed.

A role for ferritin in the antioxidant system in coffee cell cultures

Iron (Fe) is an essential nutrient for plants, but it can generate oxidative stress at high concentrations. In this study, Coffea arabica L. cell suspension cultures were exposed to excess Fe (60 and 240 mu M) to investigate changes in the gene expression of ferritin and antioxidant enzymes. Iron content accumulated during cell growth, and Western blot analysis showed an increase of ferritin in cells treated with Fe. The expression of two ferritin genes retrieved from the Brazilian coffee EST database was studied. CaFER1, but not CaFER2, transcripts were induced by Fe exposure. Phylogenetic analysis revealed that CaFER1 is not similar to CaFER2 or to any ferritin that has been characterised in detail. The increase in ferritin gene expression was accompanied by an increase in the activity of antioxidant enzymes. Superoxide dismutase, guaiacol peroxidase, catalase, and glutathione reductase activities increased in cells grown in the presence of excess Fe, especially at 60 mu M, while the activity of glutathione S-transferase decreased. These data suggest that Fe induces oxidative stress in coffee cell suspension cultures and that ferritin participates in the antioxidant system to protect cells against oxidative damage. Thus, cellular Fe concentrations must be finely regulated to avoid cellular damage most likely caused by increased oxidative stress induced by Fe. However, transcriptional analyses indicate that ferritin genes are differentially controlled, as only CaFER1 expression was responsive to Fe treatment.

Avaliação da proteção conferida pela via de sinalização do Nrf2 num modelo experimental de sobrecarga de ferro in vivo

O ferro é encontrado em praticamente todos os seres vivos, sendo um cofator para proteínas que desempenham funções essenciais à vida. Nos mamíferos, a maioria do ferro está incorporada na hemoglobina ou armazenado no fígado, ligado à ferritina. É absorvido pelos enterócitos, sendo a principal forma de controlo dos seus níveis. A sobrecarga de ferro pode levar a hemocromatose, podendo ser tóxica para vários órgãos. O fator de transcrição Nrf2 é importante na ativação de genes citoprotetores em situações de stress oxidativo/eletrofílico, colocando-se a hipótese de que poderá estar envolvido na resposta à progressão de doença devido à sobrecarga de ferro. Com o objetivo de determinar se a via do Nrf2 representa uma proteção contra a toxicidade do ferro a nível hepático, foram realizadas duas experiências nas quais murganhos C57BL/6 (B6) e Nrf2-/- machos foram alimentados com dieta standard ou com dieta enriquecida em ferro carbonilo (FeC) (0,5% ou 2,0%). Os resultados demonstram sobrecarga de ferro nos animais que receberam dieta enriquecida, sendo que os que receberam FeC 2,0% apresentaram níveis mais elevados de ferro hepático e sérico, bem como da saturação da transferrina. Os murganhos Nrf2-/- são mais suscetíveis a esta acumulação, mostrando evidências patológicas mais graves, nomeadamente necrose hepatocítica e infiltração de células inflamatórias. A deleção do Nrf2 associado a uma dieta suplementada com FeC 2,0% parece não ser suficiente para o desenvolvimento de fibrose hepática. O estudo da expressão de genes e proteínas do metabolismo do ferro mostrou que os animais B6 e Nrf2-/- são igualmente capazes de responder à sobrecarga de ferro, sugerindo que a sua diferente suscetibilidade à toxicidade do ferro não se deverá a uma regulação ineficiente da homeostasia do Fe. A dieta com FeC 2,0% aumentou a expressão de dois genes alvo do Nrf2, Nqo1 e Gsta1, o que não se verificou com os genes e proteínas GCLC e GCLM. A expressão de genes pró-inflamatórios não mostrou evidências de inflamação nestes animais. Foi demonstrado que os animais Nrf2-/- são mais suscetíveis à toxicidade do ferro, concluindo-se que a via do Nrf2 é ativada em resposta a uma dieta contendo quantidades excessivas de FeC e que confere proteção contra a acumulação de ferro em murganhos B6.

Preclinical Pharmacokinetics, Pharmacodynamics and Safety of Sucroferric Oxyhydroxide.

Sucroferric oxyhydroxide (VELPHORO(®)) is a polynuclear iron-based phosphate binder recently approved for the treatment of hyperphosphataemia in patients with chronic kidney disease (CKD). As a number of the available phosphate binders do not provide the optimal combination of good efficacy, adequate tolerability and low pill burden, sucroferric oxyhydroxide constitutes a promising alternative. Among the attributes of an ideal phosphate binder is minimal absorption and, hence, low risk of systemic toxicity. Accordingly, the iron-releasing properties and absorption, distribution, metabolism and excretion (ADME) profile of sucroferric oxyhydroxide, as well as the possibility of iron accumulation and toxicity, were investigated in a series of preclinical studies. The effect of sucroferric oxyhydroxide on the progression of vascular calcification was also investigated. Sucroferric oxyhydroxide exhibited a high phosphate-binding capacity and low iron-releasing properties across the physiological pH range found in the gastrointestinal tract. In the ADME studies, uptake of (59)Fe-radiolabelled sucroferric oxyhydroxide was low in rats and dogs (<1% from a 50 mg Fe/kg bodyweight dose), with the majority of absorbed iron located in red blood cells. Long-term (up to 2 years) administration of sucroferric oxyhydroxide in rats and dogs was associated with modest increases in tissue iron levels and no iron toxicity. Moreoever, in uraemic rats, sucroferric oxyhydroxide was associated with reduced progression of vascular calcification compared with calcium carbonate. In conclusion, sucroferric oxyhydroxide offers a new option for the treatment of hyperphosphataemia, with a high phosphate-binding capacity, minimal iron release, and low potential for iron accumulation and toxicity.

Bleaching effectiveness, hydrogen peroxide diffusion, and cytotoxicity of a chemically activated bleaching gel

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Non-coding keratin variants associate with liver fibrosis progression in patients with hemochromatosis

Background Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis. Methods The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed. Results We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury. Conclusion In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.

In vitro toxicity screening of silica-coated superparamagnetic iron oxide nanoparticles in glial cells

Nanotechnology industry is progressing with prospects of substantial benefits to economics and science. Superparamagnetic iron oxide nanoparticles (ION) have been showing excellent magnetic properties, biocompatibility and biodegradability, broadening their potential applications and importance in the biomedical field

Paracetamol degradation intermediates and toxicity during photo-Fenton treatment using different iron species

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Iron chelating-mediated antioxidant activity of Plectranthus barbatus extract on mitochondria

Plectranthus barbatus Andrews (Lamiaceae) is a popular medicinal plant used to treat gastrointestinal and hepatic ailments. In this work, we assessed the antioxidant activity of the aqueous extract of P. barbatus leaves on Fe(2+)-citrate-mediated membrane lipid peroxidation in isolated rat liver mitochondria, as well in non-mitochondrial systems: DPPH reduction, (center dot)OH scavenging activity, and iron chelation by prevention of formation of the Fe(2+)-bathophenanthroline disulfonic acid (BPS) complex. Within all the tested concentrations (15-75 mu g/ml), P. barbatus extract presented significant free radical-scavenging activity (IC(50) = 35.8 +/- 0.27 mu g/ml in the DPPH: assay and IC(50) = 69.1 +/- 0.73 mu g/ml in the (center dot)OH assay) and chelated iron (IC(50) = 30.4 +/- 3.31 mu g/ml). Over the same concentration range, the plant extract protected mitochondria against Fe(2+)/citrate-mediated swelling and malondialdehyde production, a property that persisted even after simulation of its passage through the digestive tract. These effects could be attributed to the phenolic compounds, nepetoidin - caffeic acid esters, present in the extract. Therefore, P. barbatus extract prevents mitochondrial membrane lipid peroxidation, probably by chelation of iron, revealing potential applicability as a therapeutic source of molecules against diseases involving mitochondrial iron overload. (C) 2010 Elsevier Ltd. All rights reserved.

In vitro study of CD133 human stem cells labeled with superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) are applied in stem cell labeling because of their high magnetic susceptibility as compared with ordinary paramagnetic species, their low toxicity, and their ease of magnetic manipulation. The present work is the study of CD133(+) stem cell labeling by SPIONs coupled to a specific antibody (AC133), resulting in the antigenic labeling of the CD133+ stem cell, and a method was developed for the quantification of the SPION content per cell, necessary for molecular imaging optimization. Flow cytometry analysis established the efficiency of the selection process and helped determine that the CD133 cells selected by chromatographic affinity express the transmembrane glycoprotein CD133. The presence of antibodies coupled to the SPION, expressed in the cell membrane, was observed by transmission electron microscopy. Quantification of the SPION concentration in the marked cells using the ferromagnetic resonance technique resulted in a value of 1.70 x 10 (13) mol iron (9.5 pg) or 7.0 x 10 (6) nanoparticles per cell ( the measurement was carried out in a volume of 2 mu L containing about 6.16 x 10 5 pg iron, equivalent to 4.5 x 10 (11) SPIONs). (c) 2008 Elsevier Inc. All rights reserved.

Iron Overload Favors the Elimination of Leishmania

Iron plays a central role in host-parasite interactions, since both intervenients need iron for survival and growth, but are sensitive to iron-mediated toxicity. The host’s iron overload is often associated with susceptibility to infection. However, it has been previously reported that iron overload prevented the growth of Leishmania major, an agent of cutaneous leishmaniasis, in BALB/c mice. In order to further clarify the impact of iron modulation on the growth of Leishmania in vivo, we studied the effects of iron supplementation or deprivation on the growth of L. infantum, the causative agent of Mediterranean visceral leishmaniasis, in the mouse model. We found that dietary iron deficiency did not affect the protozoan growth, whereas iron overload decreased its replication in the liver and spleen of a susceptible mouse strain. The fact that the iron-induced inhibitory effect could not be seen in mice deficient in NADPH dependent oxidase or nitric oxide synthase 2 suggests that iron eliminates L. infantum in vivo through the interaction with reactive oxygen and nitrogen species. Iron overload did not significantly alter the mouse adaptive immune response against L. infantum. Furthermore, the inhibitory action of iron towards L. infantum was also observed, in a dose dependent manner, in axenic cultures of promastigotes and amastigotes. Importantly, high iron concentrations were needed to achieve such effects. In conclusion, externally added iron synergizes with the host’s oxidative mechanisms of defense in eliminating L. infantum from mouse tissues. Additionally, the direct toxicity of iron against Leishmania suggests a potential use of this metal as a therapeutic tool or the further exploration of iron anti-parasitic mechanisms for the design of new drugs.

Iron Metabolism: From Health to Disease

Background Iron is vital for almost all living organisms by participating in a wide range of metabolic processes. However, iron concentration in body tissues must be tightly regulated since excessive iron may lead to microbial infections or cause tissue damage. Disorders of iron metabolism are among the most common human diseases and cover several conditions with varied clinical manifestations. Methods An extensive literature review on the basic aspects of iron metabolism was performed, and the most recent findings on this field were highlighted as well. Results New insights on iron metabolism have shed light into its real complexity, and its role in both healthy and pathological states has been recognized. Important discoveries about the iron regulatory machine and imbalances in its regulation have been made, which may lead in a near future to the development of new therapeutic strategies against iron disorders. Besides, the toxicity of free iron and its association with several pathologies has been addressed, although it requires further investigations. Conclusion This review will provide students in the fields of biochemistry and health sciences a brief and clear overview of iron physiology and toxicity, as well as imbalances in the iron homeostasis and associated pathological conditions.