RESUMEN

Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O2-at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H2O2 at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H2O2 was essentially produced by disproportionation of O2-, which constitutes the precursor of H2O2. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O2- and 0.63 mol of H2O2/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O2 to O2-, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H202 generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H2O2 generation. The efficiency of added quinones as peroxide generators decreased in the order Q1 > Q0 > Q2 > Q6 = Q10, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymati- cally by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O2- and H2O2 during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q0H2), benzoquinol, duroquinol and menadiol generated O2-with k3 values of 0.1 to 1.4 M-1 s-1 and H2O2 with k4 values of 0.009 to 4.3 m-1·s-1.

Asunto(s)

Complejo I de Transporte de Electrón , Superóxidos , Animales , Bovinos , Complejo I de Transporte de Electrón/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Peróxido de Hidrógeno/metabolismo , Mitocondrias Cardíacas/metabolismo , NAD/metabolismo , Oxígeno/metabolismo , Quinonas , Superóxidos/metabolismo , Ubiquinona/metabolismo

RESUMEN

In order to study the in vitro effect of flavan-3-ol (+)-catechin on the enzymatic activities of mitochondrial complex I and nitric oxide synthase (mtNOS), as well as the consequences on the membrane potential and H2O2 production rate, isolated mitochondria from rat heart were exposed to 3 nM to 100 µM (+)-catechin. NADH-Q1 reductase (complex I) and mtNOS activities were inhibited 25% and 50%, respectively, by the addition of 10 nM (+)-catechin to the reaction medium. Moreover, in the nM range, (+)-catechin decreased state 4 mitochondrial membrane potential by about 10 mV, but failed to change the membrane potential measured in the presence of ADP. (+)-Catechin (10 nM) inhibited not only complex I activity, but also the H2O2 production rate (35%) sustained by malate-glutamate, in accordance with the decrease observed in mitochondrial membrane potential. Considering (+)-catechin concentrations lower than 10 nM, linear and positive correlations were obtained between mitochondrial complex I activity and either NO (r2 = 0.973) or H2O2 production rates (r2 = 0.958), suggesting a functional association among these parameters. Altogether, the results indicate that (+)-catechin, at nM concentrations, inhibits mitochondrial complex I activity, leading to membrane potential decline and consequently to reduction in H2O2 and NO production rates. The decrease in mtNOS activity could also be a consequence of the direct action of (+)-catechin on the NOS structure, this effect being in accordance with the functional interaction between complex I and mtNOS, as previously reported.

Asunto(s)

Catequina/farmacología , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Corazón/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Animales , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Femenino , Cinética , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Miocardio/química , Miocardio/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/química , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

Activation of ERK1/2 implies the phosphorylation of tyrosine (pTyr) and threonine (pThr) by MEK1/2; both reactions were thought to be cytoplasmic, promoting ERK to reach the nucleus where it activates several transcription factors. In addition, H2O2 concentrations are known to modulate ERK intracellular translocation, which impacts on cellular proliferation. In this context, the objective of this work was to study the sequence of ERK phosphorylation under two redox conditions and to analyze a putative mitochondrial contribution to this process, in LP07 murine lung cells. A time-course of H2O2 administration was used and ERK phosphorylation was analyzed in cytosol, mitochondria and nuclei. At 1µM H2O2, a proliferative redox stimulus, immunoblot revealed a fast and transient increase in cytosol pTyr and a sustained increase in mitochondrial pTyr content. The detection for pThr/pTyrERK (2pERK) showed in cytosol a marked increase at 5 minutes with a fast dephosphorylation after that time, for both H2O2 concentrations. However, at 50 µM H2O2, an anti-proliferative condition, 2pERK was gradually retained in mitochondria. Interestingly, these results were confirmed by in vivo experiments using mice treated with a highly oxidizing agent [H2O2]. By the use of two ERK2 mutant constructions, where Tyr and Thr were replaced by alanine, we confirmed that 2pERK relied almost completely on pThr183. Confocal microscopy confirmed ERK subcellular distribution dependence on the incidence of cytosolic pTyr and mitochondrial pThr at 1µM H2O2. This work shows for the first time, both in vitro and in vivo, an ERK cycle involving a cross-talk between cytosol and mitochondria phosphorylation events, which may play a significant role in cell cycle progression, proliferation or differentiation under two different redox conditions.

Asunto(s)

Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Pulmón/metabolismo , Treonina/metabolismo , Tirosina/metabolismo , Animales , Línea Celular , Factor de Crecimiento Epidérmico/farmacología , Peróxido de Hidrógeno/metabolismo , Pulmón/citología , Masculino , Ratones , Mitocondrias/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Oxidación-Reducción , Fosforilación , Fracciones Subcelulares/metabolismo

RESUMEN

This study, in an experimental model of type I Diabetes Mellitus in rats, deals with the mitochondrial production rates and steady-state concentrations of H2O2 and NO, and ATP levels as part of a network of signaling molecules involved in heart mitochondrial biogenesis. Sustained hyperglycemia leads to a cardiac compromise against a work overload, in the absence of changes in resting cardiac performance and of heart hypertrophy. Diabetes was induced in male Wistar rats by a single dose of Streptozotocin (STZ, 60mg × kg-1, ip.). After 28 days of STZ-injection, rats were sacrificed and hearts were isolated. The mitochondrial mass (mg mitochondrial protein × g heart-1), determined through cytochrome oxidase activity ratio, was 47% higher in heart from diabetic than from control animals. Stereological analysis of cardiac tissue microphotographs showed an increase in the cytosolic volume occupied by mitochondria (30%) and in the number of mitochondria per unit area (52%), and a decrease in the mean area of each mitochondrion (23%) in diabetic respect to control rats. Additionally, an enhancement (76%) in PGC-1α expression was observed in cardiac tissue of diabetic animals. Moreover, heart mitochondrial H2O2 (127%) and NO (23%) productions and mtNOS expression (132%) were higher, while mitochondrial ATP production rate was lower (~ 40%), concomitantly with a partial-mitochondrial depolarization, in diabetic than in control rats. Changes in mitochondrial H2O2 and NO steady-state concentrations and an imbalance between cellular energy demand and mitochondrial energy transduction could be involved in the signaling pathways that lead to the novo synthesis of mitochondria. However, this compensatory mechanism triggered to restore the mitochondrial and tissue normal activities, did not lead to competent mitochondria capable of supplying the energetic demands in diabetic pathological conditions.

Asunto(s)

Adenosina Trifosfato/metabolismo , Diabetes Mellitus Experimental/metabolismo , Peróxido de Hidrógeno/metabolismo , Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Óxido Nítrico/metabolismo , Animales , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Expresión Génica , Masculino , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias Cardíacas/patología , Miocardio/patología , Biogénesis de Organelos , Tamaño de los Orgánulos , Estrés Oxidativo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Ratas , Ratas Wistar , Estreptozocina

RESUMEN

Rat liver mitochondria (1.5-2.1mg protein·mL-1) supplemented with either 25 and 100µM Cu2+ or 100 and 500µM Fe3+ show inhibition of active respiration (O2 consumption in state 3) and increased phospholipid peroxidation . Liver mitochondria were supplemented with the antioxidants reduced glutathione, N-acetylcysteine or butylated hydroxitoluene, to evaluate their effects on the above-mentioned alterations. Although the mitochondrial dysfunction is clearly associated to phospholipid peroxidation, the different responses to antioxidant supplementation indicate that the metal ions have differences in their mechanisms of toxicity. Mitochondrial phospholipid peroxidation through the formation of hydroxyl radical by a Fenton/Haber-Weiss mechanism seems to precede the respiratory inhibition and to be the main fact in Fe-induced mitochondrial dysfunction. In the case of Cu2+, it seems that the ion oxidizes glutathione, and low molecular weight protein thiol groups in a direct reaction, as part of its intracellular redox cycling. The processes involving phospholipid peroxidation, protein oxidation and mitochondrial respiratory inhibition characterize a redox dyshomeostatic situation that ultimately leads to cell death. However, Cu2+ exposure involves an additional, yet unidentified, toxic event as previous reduction of the metal with N-acetylcysteine has only a minor effect in preventing the mitochondrial damage.

Asunto(s)

Antioxidantes/farmacología , Respiración de la Célula/efectos de los fármacos , Cobre/farmacología , Hierro/fisiología , Peroxidación de Lípido/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Animales , Cobre/química , Radicales Libres/metabolismo , Iones/farmacología , Hierro/química , Masculino , Modelos Biológicos , Fosfolípidos/metabolismo , Ratas

RESUMEN

Transition from compensated to decompensated left ventricular hypertrophy (LVH) is accompanied by functional and structural changes. Here, the aim was to evaluate dystrophin expression in murine models and human subjects with LVH by transverse aortic constriction (TAC) and aortic stenosis (AS), respectively. We determined whether doxycycline (Doxy) prevented dystrophin expression and myocardial stiffness in mice. Additionally, ventricular function recovery was evaluated in patients 1 year after surgery. Mice were subjected to TAC and monitored for 3 weeks. A second group received Doxy treatment after TAC. Patients with AS were stratified by normal left ventricular end-diastolic wall stress (LVEDWS) and high LVEDWS, and groups were compared. In mice, LVH decreased inotropism and increased myocardial stiffness associated with a dystrophin breakdown and a decreased mitochondrial O2 uptake (MitoMVO2). These alterations were attenuated by Doxy. Patients with high LVEDWS showed similar results to those observed in mice. A correlation between dystrophin and myocardial stiffness was observed in both mice and humans. Systolic function at 1 year post-surgery was only recovered in the normal-LVEDWS group. In summary, mice and humans present diastolic dysfunction associated with dystrophin degradation. The recovery of ventricular function was observed only in patients with normal LVEDWS and without dystrophin degradation. In mice, Doxy improved MitoMVO2. Based on our results it is concluded that the LVH with high LVEDWS is associated to a degradation of dystrophin and increase of myocardial stiffness. At least in a murine model these alterations were attenuated after the administration of a matrix metalloprotease inhibitor.

Asunto(s)

Distrofina/deficiencia , Hipertrofia Ventricular Izquierda/metabolismo , Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Proteolisis , Animales , Modelos Animales de Enfermedad , Doxiciclina/efectos adversos , Doxiciclina/farmacología , Humanos , Hipertrofia Ventricular Izquierda/inducido químicamente , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/patología , Masculino , Ratones , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/patología , Miocardio/patología

RESUMEN

Several oxidative stress markers and liver oxygen consumption were measured in different tissues of the marine fish Trichiurus lepturus in late summer and late winter, as well as in juveniles and adult females. Oxygen consumption in liver, superoxide dismutase (SOD) and catalase (CAT) activity in liver, red cells, lens and roe, vitamin E, ubiquinol10, ß-carotene in liver, red cells, and roe, as well as contents of reduced glutathione (GSH) and lipoperoxidation (TBARS) in red cells were evaluated. Regarding ontogeny, compared to adult fish, juveniles showed significant higher SOD activity in liver and lens, as well as higher liver contents of vitamin E. In contrast, adult females showed higher contents of vitamin E in roe, ubiquinol10 in liver and roe, and higher GSH levels in red cells, while the other markers remained unchanged. Regarding seasonal changes, no differences were detected in adult females for liver CAT and ubiquinol10, CAT in roe, vitamin E in roe and in red cells, liver and red cell ubiquinol10, and in GSH in red cells. However, and coinciding with the spawning period of late summer, liver oxygen consumption, SOD and CAT activity and ubiquinol10 contents in roe and SOD activity in red cells, and red cell TBARS contents were higher compared to late winter. These temporal antioxidant adjustments of Trichiurus lepturus seem to be parallel to the higher oxygen consumption typical of juvenile forms and also to the intense spawning and foraging activities of adult females in late summer.

Asunto(s)

Proteínas de Peces/metabolismo , Peces/fisiología , Peroxidación de Lípido , Hígado/metabolismo , Morfogénesis , Estrés Oxidativo , Oxidorreductasas/metabolismo , Animales , Islas del Atlántico , Océano Atlántico , Conducta Animal , Biomarcadores/sangre , Biomarcadores/metabolismo , Brasil , Eritrocitos/enzimología , Eritrocitos/metabolismo , Conducta Alimentaria , Femenino , Peces/sangre , Peces/crecimiento & desarrollo , Glutatión/sangre , Hígado/enzimología , Hígado/crecimiento & desarrollo , Óvulo/enzimología , Óvulo/metabolismo , Oxidorreductasas/sangre , Consumo de Oxígeno , Reproducción , Estaciones del Año , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo

RESUMEN

Intercellular communication is one of the most important events in cell population behavior. In the last decade, tunneling nanotubes (TNTs) have been recognized as a new form of long distance intercellular connection. TNT function is to allow molecular and subcellular structure exchange between neighboring cells via the transfer of molecules and organelles such as calcium ions, prions, viral and bacterial pathogens, small lysosomes and mitochondria. New findings support the concept that mesenchymal stem cells (MSCs) can affect cell microenvironment by the release of soluble factors or the transfer of cellular components to neighboring cells, in a way which significantly contributes to cell regulation and tissue repair, although the underlying mechanisms remain poorly understood. MSCs have many advantages for their implementation in regenerative medicine. The TNTs in these cell types are heterogeneous in both structure and function, probably due to their highly dynamic behavior. In this work we report an extensive and detailed description of types, structure, components, dynamics and functionality of the TNTs bridging neighboring human umbilical cord MSCs obtained from Wharton"s jelly. Characterization studies were carried out through phase contrast, fluorescence, electron microscopy and time lapse images with the aim of describing cells suitable for an eventual regenerative medicine.

Asunto(s)

Comunicación Celular , Células Madre Mesenquimatosas/metabolismo , Nanotubos/química , Humanos , Células Madre Mesenquimatosas/citología

RESUMEN

Increased copper (Cu) and iron (Fe) levels in liver and brain are associated to oxidative stress and damage with increased phospholipid oxidation process. The aim of this work was to assess the toxic effects of Cu2+ and Fe3+ addition to rat liver mitochondria by determining mitochondrial respiration in states 3 (active respiration) and 4 (resting respiration), and phospholipid peroxidation. Both, Cu2+ and Fe3+ produced decreases in O2 consumption in a concentration-dependent manner in active state 3: both ions by 42% with malate-glutamate as complex I substrate (concentration for half maximal response (C50) 60µM Cu2+ and 1.25mM Fe3+), and with succinate as complex II substrate: 64-69% with C50 of 50µM Cu2+ and with C50 of 1.25mM of Fe3+. Respiratory control decreased with Cu2+ (C50 50µM) and Fe3+ (C50 1.25-1-75mM) with both substrates. Cu2+ produced a 2-fold increase and Fe3+ a 5-fold increase of thiobarbituric acid-reactive substances (TBARS) content from 25µM Cu2+ (C50 40µM) and from 100µM Fe3+ (C50 1.75mM). Supplementations with Cu2+ and Fe3+ ions induce mitochondrial dysfunction with phospholipid peroxidation in rat liver mitochondria. Although is proved that a Fenton/Haber Weiss mechanism of oxidative damage occurs in metal-ion induced mitochondrial toxicity, slightly different responses to the metal ions suggest some differences in the mechanism of intracellular toxicity. The decreased rates of mitochondrial respiration and the alteration of mitochondrial function by phospholipid and protein oxidations lead to mitochondrial dysfunction, cellular dyshomeostasis and cell death.

Asunto(s)

Cobre/farmacología , Hierro/farmacología , Peroxidación de Lípido/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Fosfolípidos/metabolismo , Animales , Complejo I de Transporte de Electrón/metabolismo , Masculino , Mitocondrias Hepáticas/patología , Proteínas Mitocondriales/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

Diabetes is a chronic disease associated to a cardiac contractile dysfunction that is not attributable to underlying coronary artery disease or hypertension, and could be consequence of a progressive deterioration of mitochondrial function. We hypothesized that impaired mitochondrial function precedes Diabetic Cardiomyopathy. Thus, the aim of this work was to study the cardiac performance and heart mitochondrial function of diabetic rats, using an experimental model of type I Diabetes. Rats were sacrificed after 28days of Streptozotocin injection (STZ, 60mgkg-1, ip.). Heart O2 consumption was declined, mainly due to the impairment of mitochondrial O2 uptake. The mitochondrial dysfunction observed in diabetic animals included the reduction of state 3 respiration (22%), the decline of ADP/O ratio (∼15%) and the decrease of the respiratory complexes activities (22-26%). An enhancement in mitochondrial H2O2 (127%) and NO (23%) production rates and in tyrosine nitration (58%) were observed in heart of diabetic rats, with a decrease in Mn-SOD activity (∼50%). Moreover, a decrease in contractile response (38%), inotropic (37%) and lusitropic (58%) reserves were observed in diabetic rats only after a ß-adrenergic stimulus. Therefore, in conditions of sustained hyperglycemia, heart mitochondrial O2 consumption and oxidative phosphorylation efficiency are decreased, and H2O2 and NO productions are increased, leading to a cardiac compromise against a work overload. This mitochondrial impairment was detected in the absence of heart hypertrophy and of resting cardiac performance changes, suggesting that mitochondrial dysfunction could precede the onset of diabetic cardiac failure, being H2O2, NO and ATP the molecules probably involved in mitochondrion-cytosol signalling.

Asunto(s)

Diabetes Mellitus Experimental/complicaciones , Cardiomiopatías Diabéticas/etiología , Cardiomiopatías Diabéticas/fisiopatología , Mitocondrias Cardíacas/patología , Adenosina Trifosfato/metabolismo , Animales , Peróxido de Hidrógeno/metabolismo , Óxido Nítrico/metabolismo , Ratas

RESUMEN

Thioredoxin is one of the most important cellular antioxidant systems known to date, and is responsible of maintaining the reduced state of the intracellular space. Trx-1 is a small cytosolic protein whose transcription is induced by stress. Therefore it is possible that this antioxidant plays a protective role against the oxidative stress caused by an increase of reactive oxygen species concentration, as occurs during the reperfusion after an ischemic episode. However, in addition to its antioxidant properties, it is able to activate other cytoplasmic and nuclear mediators that confer cardioprotection. It is remarkable that Trx-1 also participates in myocardial protection mechanisms such as ischemic preconditioning and postconditioning, activating proteins related to cellular survival. In this sense, it has been shown that Trx-1 inhibition abolished the preconditioning cardioprotective effect, evidenced through apoptosis and infarct size. Furthermore, ischemic postconditioning preserves Trx-1 content at reperfusion, after ischemia. However, comorbidities such as aging can modify this powerful cellular defense leading to decrease cardioprotection. Even ischemic preconditioning and postconditioning protocols performed in aged animal models failed to decrease infarct size. Therefore, the lack of success of antioxidants therapies to treat ischemic heart disease could be solved, at least in part, avoiding the damage of Trx system.

Asunto(s)

Poscondicionamiento Isquémico , Precondicionamiento Isquémico , Daño por Reperfusión Miocárdica/metabolismo , Tiorredoxinas/metabolismo , Envejecimiento/metabolismo , Envejecimiento/fisiología , Animales , Vasos Coronarios/fisiología , Corazón/fisiopatología , Humanos , Miocardio/metabolismo , Tiorredoxinas/fisiología

RESUMEN

AIM: We evaluated the effect of thioredoxin1 (Trx1) system on postischemic ventricular and mitochondrial dysfunction using transgenic mice overexpressing cardiac Trx1 and a dominant negative (DN-Trx1) mutant (C32S/C35S) of Trx1. Langendorff-perfused hearts were subjected to 15 min of ischemia followed by 30 min of reperfusion (R). We measured left ventricular developed pressure (LVDP, mmHg), left ventricular end diastolic pressure (LVEDP, mmHg), and t63 (relaxation index, msec). Mitochondrial respiration, SERCA2a, phospholamban (PLB), and phospholamban phosphorylation (p-PLB) Thr17 expression (Western blot) were also evaluated. RESULTS: At 30 min of reperfusion, Trx1 improved contractile state (LVDP: Trx1: 57.4 ± 4.9 vs. Wt: 27.1 ± 6.3 and DN-Trx1: 29.2 ± 7.1, p < 0.05); decreased myocardial stiffness (LVEDP: Wt: 24.5 ± 4.8 vs. Trx1: 11.8 ± 2.9, p < 0.05); and improved the isovolumic relaxation (t63: Wt: 63.3 ± 3.2 vs. Trx1: 51.4 ± 1.9, p < 0.05). DN-Trx1 mice aggravated the myocardial stiffness and isovolumic relaxation. Only the expression of p-PLB Thr17 increased at 1.5 min R in Wt and DN-Trx1 groups. At 30 min of reperfusion, state 3 mitochondrial O2 consumption was impaired by 13% in Wt and by 33% in DN-Trx1. ADP/O ratios for Wt and DN-Trx1 decrease by 25% and 28%, respectively; whereas the Trx1 does not change after ischemia and reperfusion (I/R). Interestingly, baseline values of complex I activity were increased in Trx1 mice; they were 24% and 47% higher than in Wt and DN-Trx1 mice, respectively (p < 0.01). INNOVATION AND CONCLUSION: These results strongly suggest that Trx1 ameliorates the myocardial effects of I/R by improving the free radical-mediated damage in cardiac and mitochondrial function, opening the possibility of new therapeutic strategies in coronary artery disease. Antioxid. Redox Signal. 25, 78-88.

Asunto(s)

Mitocondrias Cardíacas/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Aturdimiento Miocárdico/metabolismo , Tiorredoxinas/metabolismo , Disfunción Ventricular/metabolismo , Animales , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Peróxido de Hidrógeno/metabolismo , Masculino , Ratones , Contracción Miocárdica , Daño por Reperfusión Miocárdica/genética , Aturdimiento Miocárdico/genética , Consumo de Oxígeno , Tiorredoxinas/genética , Disfunción Ventricular/genética

RESUMEN

Dose- and time-dependent antioxidant responses to Fe (0-60 mg kg(-1)) and Cu overloads (0-30 mg kg(-1)) in rat brains are described by the C50 and the t1/2, the brain metal concentration and the time for half maximal oxidative responses. Brain GSH and the GSH/GSSG ratio markedly decreased after Fe and Cu treatments (50-80%) with a t1/2 of 9-10 h for GSH and of 4 h for GSH/GSSG for both metals. The GSH/GSSG ratio was the most sensitive indicator of brain oxidative stress. The decrease of GSH and the increase of in vivo chemiluminescence had similar time courses. The C50 for brain chemiluminescence, GSH and hydrophilic and lipophilic antioxidants were in similar ranges (32-36 µg Fe g(-1) brain and 10-18 µg Cu g(-1) brain), which indicated a unique free-radical mediated process for each metal. The brain concentration of hydrophilic and lipophilic antioxidants decreased after Fe and Cu loads; hydrophilic antioxidants decreased by 46-68% with a t1/2 of 10-11 h and lipophilic antioxidants decreased by 75-45% with a t1/2 of 10-12 h. Cu,Zn-SOD and CAT activities and the protein expression were adaptively increased (100-90% after Fe and Cu loads), with a t1/2 of 8-12 h. GPx-4 activity decreased after both metal loads by 73-27% with a t1/2 of 8-4 h with decreased protein expression.

Asunto(s)

Antioxidantes/metabolismo , Química Encefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Cobre/toxicidad , Hierro/toxicidad , Animales , Antioxidantes/análisis , Encéfalo/enzimología , Encéfalo/metabolismo , Masculino , Oxidorreductasas/análisis , Oxidorreductasas/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

The rat liver antioxidant response to Fe and Cu overloads (0-60mg/kg) was studied. Dose- and time-responses were determined and summarized by t1/2 and C50, the time and the liver metal content for half maximal oxidative responses. Liver GSH (reduced glutathione) and GSSG (glutathione disulfide) were determined. The GSH content and the GSH/GSSG ratio markedly decreased after Fe (58-66%) and Cu (79-81%) loads, with t1/2 of 4.0 and 2.0h. The C50 were in a similar range for all the indicators (110-124µgFe/g and 40-50µgCu/g) and suggest a unique free-radical mediated process. Hydrophilic antioxidants markedly decreased after Fe and Cu (60-75%; t1/2: 4.5 and 4.0h). Lipophilic antioxidants were also decreased (30-92%; t1/2: 7.0 and 5.5h) after Fe and Cu. Superoxide dismutase (SOD) activities (Cu,Zn-SOD and Mn-SOD) and protein expression were adaptively increased after metal overloads (Cu,Zn-SOD: t1/2: 8-8.5h and Mn-SOD: t1/2: 8.5-8.0h). Catalase activity was increased after Fe (65%; t1/2: 8.5h) and decreased after Cu (26%; t1/2: 8.0h), whereas catalase expression was increased after Fe and decreased after Cu overloads. Glutathione peroxidase activity decreased after metal loads by 22-39% with a t1/2 of 4.5h and with unchanged protein expression. GSH is the main and fastest responder antioxidant in Fe and Cu overloads. The results indicate that thiol (SH) content and antioxidant enzyme activities are central to the antioxidant defense in the oxidative stress and damage after Fe and Cu overloads.

Asunto(s)

Cobre/administración & dosificación , Hierro/administración & dosificación , Peroxidación de Lípido , Hígado/efectos de los fármacos , Estrés Oxidativo , Animales , Antioxidantes/química , Antioxidantes/metabolismo , Catalasa/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión/biosíntesis , Glutatión Peroxidasa/biosíntesis , Hígado/metabolismo , Ratas , Superóxido Dismutasa/biosíntesis

RESUMEN

This study reports on the acute brain toxicity of Fe and Cu in male Sprague-Dawley rats (200 g) that received 0 to 60 mg kg(-1) (ip) FeCl2 or CuSO4. Brain metal contents and time-responses were determined for rat survival, in situ brain chemiluminescence and phospholipid and protein oxidation products. Metal doses hyperbolically defined brain metal content. Rat survival was 91% and 60% after Fe and Cu overloads. Brain metal content increased from 35 to 114 µg of Fe per g and from 3.6 to 34 µg of Cu per g. Brain chemiluminescence (10 cps cm(-2)) increased 3 and 2 times after Fe and Cu overloads, with half maximal responses (C50) of 38 µg of Fe per g of brain and 15 µg of Cu per g of brain, and with half time responses (t1/2) of 12 h for Fe and 20 h for Cu. Phospholipid peroxidation increased by 56% and 31% with C50 of 40 µg of Fe per g and 20 µg of Cu per g and with t1/2 of 9 h and 14 h. Protein oxidation increased by 45% for Fe with a C50 of 40 µg of Fe per g and 18% for Cu with a C50 of 10 µg of Cu per g and a t1/2 of 12 h for both metals. Fe and Cu brain toxicities are likely mediated by Haber-Weiss type HO˙ formation with subsequent oxidative damage.

Asunto(s)

Encéfalo/patología , Cobre/metabolismo , Hierro/metabolismo , Animales , Masculino , Estrés Oxidativo/fisiología , Ratas , Ratas Sprague-Dawley

RESUMEN

Weanling rats fed a choline-deficient diet develop kidney oxidative damage, tubular and cortical kidney necrosis, renal failure and animal death. The effect of dietary menhaden oil was assayed on the mentioned sequence correlating oxidative stress with renal structure and function. Rats were fed ad libitum 4 different diets: (a) a choline-deficient diet with corn oil and sunflower hydrogenated oil as a source of fatty acids; (b) the same diet supplemented with choline; (c) a choline-deficient diet with menhaden oil as a source of fatty acids; and (d) the previous diet supplemented with choline. Animals were sacrificed at days 0, 2, 4 and 7. The histopathological study of the kidneys showed that renal necrosis was only observed at day 7 in choline-deficient rats receiving the vegetable oil diet, simultaneously with increased creatinine plasma levels. Homogenate chemiluminescence (BOOH-initiated chemiluminescence) and phospholipid oxidation indicate the development of oxidative stress and damage in choline-deficient rats fed vegetable oils as well as the protective effect of menhaden oil. Rats fed with the fish oil diet showed that oxidative stress and damage develop later, as compared with vegetable oil, with no morphological damage during the experimental period.

Asunto(s)

Deficiencia de Colina/tratamiento farmacológico , Colina/administración & dosificación , Suplementos Dietéticos , Aceites de Pescado/administración & dosificación , Enfermedades Renales/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Enfermedad Aguda , Animales , Deficiencia de Colina/complicaciones , Deficiencia de Colina/patología , Aceite de Maíz/administración & dosificación , Creatinina/sangre , Dieta , Riñón/efectos de los fármacos , Riñón/patología , Enfermedades Renales/etiología , Enfermedades Renales/patología , Masculino , Necrosis , Fosfolípidos/metabolismo , Aceites de Plantas/administración & dosificación , Ratas , Ratas Wistar , Aceite de Girasol

RESUMEN

The transition metals iron (Fe) and copper (Cu) are needed at low levels for normal health and at higher levels they become toxic for humans and animals. The acute liver toxicity of Fe and Cu was studied in Sprague Dawley male rats (200 g) that received ip 0-60 mg/kg FeCl(2) or 0-30 mg/kg CuSO(4). Dose and time-responses were determined for spontaneous in situ liver chemiluminescence, phospholipid lipoperoxidation, protein oxidation and lipid soluble antioxidants. The doses linearly defined the tissue content of both metals. Liver chemiluminescence increased 4 times and 2 times after Fe and Cu overloads, with half maximal responses at contents (C(50%)) of 110 µgFe/g and 42 µgCu/g liver, and with half maximal time responses (t(1/2)) of 4h for both metals. Phospholipid peroxidation increased 4 and 1.8 times with C(50%) of 118 µg Fe/g and 45 µg Cu/g and with t(1/2) of 7h and 8h. Protein oxidation increased 1.6 times for Fe with C(50%) at 113 µg Fe/g and 1.2 times for Cu with 50 µg Cu/g and t(1/2) of 4h and 5h respectively. The accumulation of Fe and Cu in liver enhanced the rate of free radical reactions and produced oxidative damage. A similar free radical-mediated process, through the formation HO(•) and RO(•) by a Fenton-like homolytic scission of H(2)O(2) and ROOH, seems to operate as the chemical mechanism for the liver toxicity of both metals.

Asunto(s)

Cobre/toxicidad , Hierro/toxicidad , Hígado/efectos de los fármacos , Estrés Oxidativo , Animales , Semivida , Peroxidación de Lípido , Hígado/metabolismo , Luminiscencia , Masculino , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley

RESUMEN

Acute endotoxemia (LPS, 10 mg/kg ip, Sprague Dawley rats, 45 days old, 180 g) decreased the O2 consumption of rat heart (1 mm³ tissue cubes) by 33% (from 4.69 to 3.11 µmol O2/min. g tissue). Mitochondrial O2 consumption and complex I activity were also decreased by 27% and 29%, respectively. Impaired respiration was associated to decreased ATP synthesis (from 417 to 168 nmol/min. mg protein) and ATP content (from 5.40 to 4.18 nmol ATP/mg protein), without affecting mitochondrial membrane potential. This scenario is accompanied by an increased production of O2·â» and H2O2 due to complex I inhibition. The increased NO production, as shown by 38% increased mtNOS biochemical activity and 31% increased mtNOS functional activity, is expected to fuel an increased ONOO⁻ generation that is considered relevant in terms of the biochemical mechanism. Heart mitochondrial bioenergetic dysfunction with decreased O2 uptake, ATP production and contents may indicate that preservation of mitochondrial function will prevent heart failure in endotoxemia.

Asunto(s)

Adenosina Trifosfato/biosíntesis , Complejo I de Transporte de Electrón/metabolismo , Endotoxemia/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias Cardíacas/metabolismo , Consumo de Oxígeno , Animales , Transporte de Electrón , Endotoxemia/complicaciones , Endotoxemia/patología , Femenino , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Peróxido de Hidrógeno/metabolismo , Mitocondrias Cardíacas/patología , Óxido Nítrico/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

Hemorrhage (H) is associated with a left ventricular (LV) dysfunction. However, the diastolic function has not been studied in detail. The main goal was to assess the diastolic function both during and 120 min after bleeding, in the absence and in the presence of L-NAME. Also, the changes in mRNA and protein expression of nitric oxide synthase (NOS) isoforms were determined. New Zealand rabbits were divided into three groups: Sham group, H group (hemorrhage 20% blood volume), and H L-NAME group (hemorrhage treated with L-NAME). We evaluated systolic and diastolic ventricular functions in vivo and in vitro (Langendorff technique). Hemodynamic parameters and LV function were measured before, during, and at 120 min after bleeding. We analyzed the isovolumic relaxation using t ½ in vivo (closed chest). After that, hearts were excised and perfused in vitro to measure myocardial stiffness. Samples were frozen to measure NOS mRNA and protein expression. The t½ increased during bleeding and returned to basal values 120 min after bleeding. L-NAME blunted this effect. Data from the H group revealed a shift to the left in the LV end diastolic pressure-volume curve at 120 min after bleeding, which was blocked by L-NAME. iNOS and nNOS protein expression and mRNA levels increased at 120 min after the hemorrhage. Acute hemorrhage induces early and transient isovolumic relaxation impairment and an increase in myocardial stiffness 120 min after bleeding. L-NAME blunted the LV dysfunction, suggesting that NO modulates ventricular function through iNOS and nNOS isoforms.

Asunto(s)

Diástole , Choque Hemorrágico/fisiopatología , Disfunción Ventricular Izquierda/tratamiento farmacológico , Animales , Diástole/efectos de los fármacos , Diástole/fisiología , Corazón/fisiopatología , Hemorragia , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo II , Óxidos de Nitrógeno , Conejos , Choque Hemorrágico/complicaciones , Disfunción Ventricular Izquierda/enzimología , Disfunción Ventricular Izquierda/etiología

RESUMEN

Isolated rabbit hearts were exposed to ischemia (I; 15 min) and reperfusion (R; 5-30 min) in a model of stunned myocardium. I/R decreased left-ventricle O(2) consumption (46%) and malate-glutamate-supported mitochondrial state 3 respiration (32%). Activity of complex I was 28% lower after I/R. The pattern observed for the decline in complex I activity was also observed for the reduction in mitochondrial nitric oxide synthase (mtNOS) biochemical (28%) and functional (50%) activities, in accordance with the reported physical and functional interactions between complex I and mtNOS. Malate-glutamate-supported state 4 H(2)O(2) production was increased by 78% after I/R. Rabbit heart Mn-SOD concentration in the mitochondrial matrix (7.4±0.7 µM) was not modified by I/R. Mitochondrial phospholipid oxidation products were increased by 42%, whereas protein oxidation was only slightly increased. I/R produced a marked (70%) enhancement in tyrosine nitration of the mitochondrial proteins. Adenosine attenuated postischemic ventricular dysfunction and protected the heart from the declines in O(2) consumption and in complex I and mtNOS activities and from the enhancement of mitochondrial phospholipid oxidation. Rabbit myocardial stunning is associated with a condition of dysfunctional mitochondria named "complex I syndrome." The beneficial effect of adenosine could be attributed to a better regulation of intracellular cardiomyocyte Ca(2+) concentration.

Asunto(s)

Adenosina/administración & dosificación , Complejo I de Transporte de Electrón/metabolismo , Mitocondrias Cardíacas/metabolismo , Aturdimiento Miocárdico/metabolismo , Daño por Reperfusión/metabolismo , Animales , Respiración de la Célula/efectos de los fármacos , Modelos Animales de Enfermedad , Ventrículos Cardíacos/patología , Peroxidación de Lípido , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/patología , Aturdimiento Miocárdico/tratamiento farmacológico , Aturdimiento Miocárdico/patología , Óxido Nítrico Sintasa/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Conejos , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/patología , Superóxido Dismutasa/metabolismo