Sobrecarga de hierro : efecto sobre el estrés oxidativo en cerebro de rata

Fil: Piloni, Natacha Estefanía. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, ArgentinaEl objetivo general de esta tesis fue estudiar el efecto de la sobrecarga de Fe en cerebro de rata. Se utilizó un modelo agudo y uno subcrónico de exposición a Fe-dextrán. Se estudiaron los mecanismos vinculados al metabolismo oxidativo y el establecimiento de una posible respuesta hormética generada por la administración de otros agentes estresantes.\nLa sobrecarga aguda de Fe aumentó el contenido de Fe total en el cerebro de rata a las 6 h de administración de la dosis de Fe, que se vio reflejado en la corteza, el hipocampo y mayoritariamente en el cuerpo estriado. Este incremento llevó a un aumento en cerebro entero en el contenido de LIP y de las ROS que desencadenaron un estado de estrés oxidativo. Aunque se observó una disminución en la integridad de ADN nuclear y mitocondrial, el estrés oxidativo generó el aumento de la activación del factor NF-?B y en la actividad de la enzima CAT, sin la producción de un daño significativo en cerebro. El incremento en la peroxidación lipídica observado en la corteza, acompañado de una respuesta antioxidante, puso en evidencia la mayor susceptibilidad a nivel lipofílico de este área cerebral y su capacidad para contrarrestar los efectos estresantes generados por la administración del Fe.\nLa sobrecarga subcrónica de Fe incrementó el contenido de Fe en cerebro entero a las 2 h luego de la 6ta dosis, en comparación con los valores obtenidos en animales no tratados, e incrementó el contenido de LIP a partir de las 2 h pi. Luego de la 6ta dosis de Fe-dextrán, se desencadenó un estado de estrés oxidativo y se observó un incremento en la actividad de SOD y CAT a las 2 y 8 h, respectivamente. El porcentaje de unión del factor NF-?B al ADN no se modificó en el período estudiado indicando el gatillado de un mecanismo de señalización del sistema de defensa antioxidante diferente al desencadenado por la sobrecarga aguda de Fe. No se observaron cambios en la integridad del ADN nuclear, aunque la integridad del ADN mitocondrial disminuyó. Estos resultados indican una adecuada capacidad del cerebro para limitar el daño debido a la sobrecarga subcrónica de Fe. El aumento en el contenido de Fe total a las 2 h luego de la administración de la 6ta dosis de Fe en cerebro total se vio reflejado en la corteza, el cuerpo estriado y mayoritariamente en el hipocampo. Se midió un incremento significativo en la actividad de CAT en la corteza. Estos resultados acentúan la mayor susceptibilidad de la corteza cerebral frente a una situación de estrés y reiteran la importancia de individualizar cada área específica del cerebro para el estudio de la respuesta frente al estrés generado por el Fe.\nLa administración simultánea de Fe y LPS en una única dosis, parece tener un efecto protector a nivel hidrofílico (evidenciado por la generación de radical ascorbilo). En cambio, la administración de LPS posterior a un tratamiento subcrónico con Fe, no da cuenta de ninguna protección adicional en este medio celular a los tiempos estudiados. Sin embargo, el tratamiento subcrónico con Fe en forma previa a la administración de CPZ, produjo un efecto protector a nivel lipofílico, que podría ser considerado como un efecto hormético. Si bien se requieren otros estudios que avalen esta hipótesis, la posibilidad de encontrar la ventana temporal en la cual el Fe genere una respuesta antioxidante capaz de contrarrestar el efecto oxidativo de drogas como la CPZ, resulta interesante no sólo desde un punto de vista clínico sino también para caracterizar los efectos del Fe a nivel mecanístico

Acute Fe-dextran treatment and redox balance in rat whole brain and cortex

An acute Fe-dextran treatment produced oxidative stress in rat brain that lead to the translocation of Nrf2 to the cell nucleus, producing the activation of genes involved in the glutathione metabolism in the cellular environment. Previous reports have shown that the acute Fe overload produced by a single injection of Fe-dextran resulted in a significant decrease in total thiol and glutathione content in rat cortex area after 6 and 8 h post injection (pi). In the whole brain, enzymatic activities of glutathione-S-transferase (GST) and glutathione peroxidase (GPx), and total thiol content were increased as compared to control tissues at 6 or 8 h pi, respectively. The aim of this study was to determine the effect of acute Fe overload on glutathione-dependent enzymatic metabolism in cortex rat brain. A single dose of 500 mg Fe-dextran/kg body weight was administrated intraperitoneally to male Sprague Dawley rats. Total brain samples or cortex area were obtained from control and treated animals after 6 or 8 h pi. Glutathione reductase (GR) was determined spectrophotometrically. Reduced glutathione (GSH), oxidized glutathione (GSSG) and malondialdehyde (MDA) content were determined by reverse phase HPLC. MDA content showed a significant increased (p<0.05) at 8 h pi in whole brain. A significant decrease in cortical GSH (p<0.05), and a significant increase in cortical GSSG (p<0.05) was observed at 8 h pi. A slight but non-significant reduction in the activity of the enzyme GR was seen at 6 and 8 h pi in brain cortex. Taking as a whole, these results suggested that the increase in the GSSG/GSH ratio could be associated to the increase in the activities of GST and GPx without any change in GR activity in brain cortex. Moreover, it seems that the alteration in the redox status caused by the Fe treatment in the cortex could contribute to the lipid peroxidation changes detected in the whole brain.Fil: Piloni, Natacha Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Robello, Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Puntarulo, Susana Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaLXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LI Reunión Anual de la Asociación Argentina de Farmacología Experimental; XXI Reunión Anual de la Sociedad Argentina de Biología; XXXI Reunión Anual de la Sociedad Argentina de Protozoología; IX Reunión Anual de la Asociación Argentina de Nanomedicinas y VI Reunión Científica Regional de la Asociación Argentina de Ciencia y Tecnología de Animales de LaboratorioMar del PlataArgentinaSociedad Argentina de Investigación ClínicaAsociación Argentina de Farmacología ExperimentalSociedad Argentina de BiologíaSociedad Argentina de ProtozoologíaAsociación Argentina de NanomedicinasAsociación Argentina de Ciencia y Tecnología de Animales de Laboratori

Update on Fe-dependent oxidative metabolism in vivo: an integrative view

Fe is essential for human life because it constitutes the required cofactor for proteins of diverse biological functions. However, the development of oxidative stress by exposure to excessive Fe, share signaling pathways with other treatments including activation of redox-sensitive factors. This study was focused on the comparison on the effects of Fe in the brain and other organs in vivo. The oxidative effects triggered by Fe overload strongly depend not only on the administration protocol, but also on the Fe-compound used, and the studied organ. In both the liver and the brain, Fe content drastically increased after Fe-dextran administration. However, the comparatively lowlipid peroxidation in the brain as compared to the liver, suggested that Fe-dependent oxidative stress might involve mechanisms of different nature. In the brain, acute and subchronic administration of Fe-dextran triggered signaling processes that lead to the prevention of injury by the participation of catalase activity as an antioxidant protection.This brief summary opens a huge range of possible points of risk, as well as opportunities, to encounter situations in which the appropriate election of the Fe management protocol could be able of allow oxidative stress to exert beneficial effects.Fil: Piloni, Natacha Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Robello, Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Bonetto, Julián Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Puntarulo, Susana Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentin

Fe overload an ascorbyl radical steady state concentration in rat brain

The one-electron oxidation of ascorbate produces the ascorbyl radical (A●), that has been proposed as a marker of oxidative stress either in vitro, or in vivo in numerous systems. The application of a simple kinetic analysis allowed us to estimate the steady state concentration of A●. These data were successfully compared to experimental values obtained in rat brain under physiological conditions and in response to stress due to sub-chronic Fe overload. The main objective of this chapter is to further explore the information presented in a previously published manuscript employing a simple kinetic model developed to estimate A● state concentration to other Fe-dependent oxidative conditions in rat brain. This analysis shows that acute Fe-overload, seems to affect A● state concentration by a more complex pathway network than sub-chronic Fe-overload. The mechanistic study presented here will help to understand the mechanisms that govern the responses of living organisms to toxicologically relevant challenges.Fil: Piloni, Natacha Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Puntarulo, Susana Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentin

Effect of Fe overload on ascorbyl radical steady state concentration in rat brain

The one-electron oxidation of ascorbate produces the ascorbyl radical (A), that has beenproposed as a marker of oxidative stress either in vitro, or in vivo in numerous systems. Theapplication of a simple kinetic analysis allowed us to estimate the steady state concentration of A.These data were successfully compared to experimental values obtained using ElectronParamagnetic Resonance (EPR) in rat brain under physiological conditions and the response tostress due to sub-chronic Fe overload. The main objective of this chapter is to further explore theinformation presented in a previously published manuscript employing a simple kinetic modeldeveloped to estimate A steady state concentration to other Fe-dependent oxidative conditions inrat brain.Fil: Piloni, Natacha Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Puntarulo, Susana Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentin

A Simple Kinetic Model to Estimate Ascorbyl Radical Steady State Concentration in Rat Central Nervous System. Effect of Subchronic Fe Overload

All biological systems, contain many antioxidants; including water-soluble compounds, such as ascorbic acid. The one-electron oxidation of ascorbate (AH¯produces the ascorbyl radical (A•), that has a relatively long lifetime compared to other species, such as hydroxyl radical, peroxyl, alkoxyl, and carbon-centered lipid free radicals. A• has been proposed as a marker of oxidative stress either in vitro, or in vivo in numerous systems. The application of a simple kinetic analysis allowed us to estimate the steady state concentration of A•. These data were successfully compared to experimental values obtained using Electron Paramagnetic Resonance (EPR) in rat brain under physiological conditions. The model was also applied to estimate the response to stress due to subchronic Fe overload.Fil: Piloni, Natacha Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Puntarulo, Susana Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentin

Iron homeostasis and oxidative balance in the context of endotoxemia

This chapter intends to contribute to a deeper understanding of the main pathways playing a role in the complex mechanisms triggered in Fe-treated organisms. It has been shown that Fe overload causes an increase, as compared to values in control animals, in serum Fe content, and in hepatic ferritin (Ft) content, Fe content in Ft, labile Fe pool (LIP), and protein carbonyl content. High levels of LIP are harmful in tissues, especially through redox damage that can lead to fibrosis. High levels of Ft can occur in several diseases including hemophagocytic lymphohistiocytosis, macrophage activation syndrome, adult-onset Still?s disease, catastrophic antiphospholipid syndrome and septic shock. Lipopolysaccharide (LPS) treatment by itself significantly decreased Fe content in serum, and increased blood NO content. Numerous studies have demonstrated the immunomodulatory effects of Ft and its association with mortality and sustained inflammatory process. A pathogenic role of Ft and Fe has even been described during SARS-CoV-2 infection. Inflammatory responses involving microglia and astrocytes contribute to the pathogenesis of neurodegenerative diseases (NDs). Ferroptosis is a Reactive Oxygen Species (ROS)-and Fe-dependent form of regulated cell death, playing a critical role in organ injury. Ferroptosis participates in the development of cardiomyopathy including cardiac hypertrophy, diabetic cardiomyopathy and doxorubicin-induced cardiotoxicity. However, the role of ferroptosis in sepsis-induced injury remains unclear. Even though inflammation is tightly linked to Fe metabolism dysregulation, it is not clear whether the dysregulation of Fe metabolism induced by brain inflammation contributes to the pathogenesis of NDs or, depending on the doses and duration of the exposure, if it leads to hormesis (beneficial effects). The protective strategy against endotoxemia of sequestering serum Fe content is not fully operative under Fe-overload conditions, even though endogenous mechanisms are able to regulate the amount of catalytically active Fe. Information linked to these aspects of LPS exposure in several organs is updated here, but further studies are necessary to wholly identify the mechanisms involved.Fil: Piloni, Natacha Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Puntarulo, Susana Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentin

Iron overload prevents oxidative damage to rat brain after chlorpromazine administration

The hypothesis tested is that Fe administration leads to a response in rat brain modulating the effects of later oxidative challenges such as chlorpromazine (CPZ) administration. Either a single dose (acute Fe overload) or 6 doses every second day (sub-chronic Fe overload) of 500 or 50 mg Fe-dextran/kg, respectively, were injected intraperitoneally (ip) to rats. A single dose of 10 mg CPZ/kg was injected ip 8 h after Fe treatment. DNA integrity was evaluated by quantitative PCR, lipid radical (LR · ) generation rate by electron paramagnetic resonance (EPR), and catalase (CAT) activity by UV spectrophotometry in isolated brains. The maximum increase in total Fe brain was detected after 6 or 2 h in the acute and sub-chronic Fe overload model, respectively. Mitochondrial and nuclear DNA integrity decreased after acute Fe overload at the time of maximal Fe content; the decrease in DNA integrity was lower after sub-chronic than after acute Fe overload. CPZ administration increased LR · generation rate in control rat brain after 1 and 2 h; however, CPZ administration after acute or sub-chronic Fe overload did not affect LR · generation rate. CPZ treatment did not affect CAT activity after 1–4 h neither in control rats nor in acute Fe-overloaded rats. However, CPZ administration to rats treated sub-chronically with Fe showed increased brain CAT activity after 2 or 4 h, as compared to control values. Fe supplementation prevented brain damage in both acute and sub-chronic models of Fe overload by selectively activating antioxidant pathways.Fil: Piloni, Natacha Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Caro, Andres A.. Hendrix College, Conway; Estados UnidosFil: Puntarulo, Susana Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentin

The role of melatonin on intracellular oxidative stress: Interaction with Fe-overload

Melatonin (MLT) is a ubiquitous compound present in bacteria andeukaryotes, which in vertebrates, is released at night from the pinealgland to induce sleep. There are also extrapineal MLT sources, since it issynthesized in most tissues and organs, such as the retina, immune cells,intestine, and bone marrow. Unlike that of pineal origin, this extrapinealMLT does not get into the bloodstream, it is not under the chronobioticeffect, and shows local antioxidant/anti-inflammatory protection abilitiesfor the tissue or organ that synthesizes it. MLT has been also related withbinding proteins, receptor distribution, and secondary effects bymetabolites. Actions via hormonal subsystems, growth factors andneurotransmission lead to further secondary effects. In the present report,we review the studies on the potential effect of MLT against Fe-overloadinducedtoxicity through its chelating effect on Fe and improvement ofantioxidant status. Up to now, signaling by MLT in the context ofantioxidant protection has been mainly related to the induction ofprotective enzymes and to the suppression of others involved in radicalgeneration, to protect cells from O2 and N2 radical molecules. MLT alsoinfluences both antioxidant enzyme activity and cellular mRNA levels forthese enzymes. Fe-overload induces severe damage to several vital organssuch as the liver, heart and bone, and thus contributes to the dysfunctionof these organs, leading to functional impairment and reduced lifeexpectancy. Moreover, it can affect the balance between pro-oxidants andantioxidants, leading to the decrease in total antioxidant capacity. MLTeffects are uncommonly widespread in several organs since its actions aremediated via membrane receptors, nuclear receptors/binding sites andreceptor-independent mechanisms, i.e., the direct scavenging of freeradicals. Up to now, it is not clear whether the protective actions of MLTare due to MLT per se, to its metabolites, to its ability to stimulateantioxidant enzymes, or due to its action of inhibiting pro-oxidativeenzymes. Taken together, this review briefly summarizes the actions ofMLT in a broad range of effects with a significant regulatory influenceover many of the protection processes against Fe-overload effects.Fil: Hernando, M. P.. Comisión Nacional de Energía Atómica; Argentina. Universidad de Morón; ArgentinaFil: Piloni, Natacha Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Cervino, Claudio Osvaldo. Universidad de Morón; ArgentinaFil: Puntarulo, Susana Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentin

Effects of acute iron overload on Nrf2-related glutathione metabolism in rat brain

Iron (Fe) overload triggers free radical production and lipid peroxidation processes that may lead to cell death (ferroptosis). The hypothesis of this work was that acute Fe-dextran treatment triggers Nrf2-mediated antioxidant regulation in rat brain involving glutathione (GSH) metabolism. Over the initial 8 h after Fe-dextran administration (single dose of 500 mg Fe-dextran/kg), total Fe, malondialdehyde (MDA) content, glutathione peroxidase (GPx), GPx-Se dependent (GPx-Se) and glutathione S-transferases (GST) activities were increased in rat whole brain. The content of GSH and the activity of glutathione reductase (GR) showed decreases (p < 0.05) after 6 and 8 h post injection in cortex. A significant increase in nuclear Nrf2 protein levels over control values was achieved after 6 h of Fe-dextran administration, while no significant differences were observed in the cytosolic fraction. Nuclear Nrf2/cytosolic Nrf2 ratios showed enhancement (p < 0.05) after 6 h of Fe overload, suggesting a greater translocation of the factor to the nucleus. No significant differences were observed in the expression of Keap1 in nuclear or cytosolic extracts. It is concluded that acute Fe overload induces oxidative stress in rat brain with the concomitant lipid peroxidation increase and GSH depletion, leading to the elevation of Nrf2-controlled GPx, GPx-Se and GST protein expression as a protective adaptive response. Further studies are required to fully comprehend the complex network of interrelated processes keeping the balance of GSH functions as chelator, antioxidant and redox buffer in the understanding of the ferroptotic and hormetic mechanisms triggered by Fe overload in brain.Fil: Piloni, Natacha Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Vargas, Romina. Universidad de Chile; ChileFil: Fernández, Virginia. Universidad de Chile; ChileFil: Videla, Luis A.. Universidad de Chile; ChileFil: Puntarulo, Susana Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentin