RESUMEN
Pyridoxal isonicotinoyl hydrazone (PIH) is an iron chelator with antioxidant activity, low toxicity and is useful in the experimental treatment of iron-overload diseases. Previous studies on x-ray diffraction have revealed that PIH also forms a complex with Cu(II). Since the main drug of choice for the treatment of Wilson's disease, d-penicillamine, causes a series of side effects, there is an urgent need for the development of alternative copper chelating agents for clinical use. These chelators must also have antioxidant activity because oxidative stress is associated with brain and liver copper-overload. In this work we tested the ability of PIH to prevent in vitro free radical formation mediated by Cu(II), ascorbate and dissolved O2. Degradation of 2-deoxyribose mediated by 10 microM Cu(II) and 3 mM ascorbate was fully inhibited by 10 microM PIH (I50 = 6 microM) or 20 microM d-penicillamine (I50 = 10 microM). The antioxidant efficiency of PIH remained unchanged with increasing concentrations (from 1 to 15 mM) of the hydroxyl radical detector molecule, 2-deoxyribose, indicating that PIH does not act as a hydroxyl scavenger. On the other hand, the efficiency of PIH (against copper-mediated 2-deoxyribose degradation and ascorbate oxidation) was inversely proportional to the Cu(II) concentration, suggesting a competition between PIH and ascorbate for complexation with Cu(lI). An almost full inhibitory effect by PIH was observed when the ratio PIH:copper was 1:1. A similar result was obtained with the measurement of copper plus ascorbate-mediated O2 uptake. Moreover, spectral studies of the copper and PIH interaction showed a peak at 455 nm and also indicated the formation of a stable Cu(II) complex with PIH with a 1:1 ratio. These data demonstrated that PIH prevents hydroxyl radical formation and oxidative damage to 2-deoxyribose by forming a complex with Cu(II) that is not reactive with ascorbate (first step of the reactions leading to hydroxyl radical formation from Cu(II), ascorbate and O2) and does not participate in Haber-Weiss reactions.
Asunto(s)
Sulfato de Cobre/antagonistas & inhibidores , Depuradores de Radicales Libres/farmacología , Radical Hidroxilo/antagonistas & inhibidores , Quelantes del Hierro/farmacología , Isoniazida/análogos & derivados , Isoniazida/farmacología , Piridoxal/análogos & derivados , Piridoxal/farmacología , Ácido Ascórbico/farmacología , Sulfato de Cobre/farmacología , Desoxirribosa/metabolismo , Radicales Libres , Radical Hidroxilo/metabolismo , Técnicas In Vitro , Cinética , Oxidación-Reducción , Oxígeno/metabolismoRESUMEN
Pyridoxal isonicotinoyl hydrazone (PIH) is able to prevent iron-mediated hydroxyl radical formation by means of iron chelation and inhibition of redox cycling of the metal. In this study, we investigated the effect of PIH on Fe(II)-citrate-mediated lipid peroxidation and damage to isolated rat liver mitochondria. Lipid peroxidation was quantified by the production of thiobarbituric acid-reactive substances (TBARS) and by antimycin A-insensitive oxygen consumption. PIH at 300 microM induced full protection against 50 microM Fe(II)-citrate-induced loss of mitochondrial transmembrane potential (deltapsi) and mitochondrial swelling. In addition, PIH prevented the Fe(II)-citrate-dependent formation of TBARS and antimycin A-insensitive oxygen consumption. The antioxidant effectiveness of 100 microM PIH (on TBARS formation and mitochondrial swelling) was greater in the presence of 20 or 50 microM Fe(II)-citrate than in the presence of 100 microM Fe(II)-citrate, suggesting that the mechanism of PIH antioxidant action is linked with its Fe(II) chelating property. Finally, PIH increased the rate of Fe(II) autoxidation by sequestering iron from the Fe(II)-citrate complex, forming a Fe(III)-PIH, complex that does not participate in Fenton-type reactions and lipid peroxidation. These results are of pharmacological relevance since PIH is a potential candidate for chelation therapy in diseases related to abnormal intracellular iron distribution and/or iron overload.
Asunto(s)
Antioxidantes/farmacología , Compuestos Férricos/antagonistas & inhibidores , Quelantes del Hierro/farmacología , Isoniazida/análogos & derivados , Isoniazida/farmacología , Peroxidación de Lípido/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Piridoxal/análogos & derivados , Piridoxal/farmacología , Animales , Quelantes/farmacología , Compuestos Férricos/farmacología , Técnicas In Vitro , Indicadores y Reactivos , Hierro/química , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Oxidación-Reducción , Consumo de Oxígeno/efectos de los fármacos , Ratas , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Iron chelating agents are essential for treating iron overload in diseases such as beta-thalassemia and are potentially useful for therapy in non-iron overload conditions, including free radical mediated tissue injury. Deferoxamine (DFO), the only drug available for iron chelation therapy, has a number of disadvantages (e.g., lack of intestinal absorption and high cost). The tridentate chelator pyridoxal isonicotinoyl hydrazone (PIH) has high iron chelation efficacy in vitro and in vivo with high selectivity and affinity for iron. It is relatively non-toxic, economical to synthesize and orally effective. We previously demonstrated that submillimolar levels of PIH and some of its analogues inhibit lipid peroxidation, ascorbate oxidation, 2-deoxyribose degradation, plasmid DNA strand breaks and 5,5-dimethylpyrroline-N-oxide (DMPO) hydroxylation mediated by either Fe(II) plus H(2)O(2) or Fe(III)-EDTA plus ascorbate. To further characterize the mechanism of PIH action, we studied the effects of PIH and some of its analogues on the degradation of 2-deoxyribose induced by Fe(III)-EDTA plus ascorbate. Compared with hydroxyl radical scavengers (DMSO, salicylate and mannitol), PIH was about two orders of magnitude more active in protecting 2-deoxyribose from degradation, which was comparable with some of its analogues and DFO. Competition experiments using two different concentrations of 2-deoxyribose (15 vs. 1.5 mM) revealed that hydroxyl radical scavengers (at 20 or 60 mM) were significantly less effective in preventing degradation of 2-deoxyribose at 15 mM than 2-deoxyribose at 1.5 mM. In contrast, 400 microM PIH was equally effective in preventing degradation of both 15 mM and 1.5 mM 2-deoxyribose. At a fixed Fe(III) concentration, increasing the concentration of ligands (either EDTA or NTA) caused a significant reduction in the protective effect of PIH towards 2-deoxyribose degradation. We also observed that PIH and DFO prevent 2-deoxyribose degradation induced by hypoxanthine, xanthine oxidase and Fe(III)-EDTA. The efficacy of PIH or DFO was inversely related to the EDTA concentration. Taken together, these results indicate that PIH (and its analogues) works by a mechanism different than the hydroxyl radical scavengers. It is likely that PIH removes Fe(III) from the chelates (either Fe(III)-EDTA or Fe(III)-NTA) and forms a Fe(III)-PIH(2) complex that does not catalyze oxyradical formation.
Asunto(s)
Ácido Ascórbico , Quelantes , Desoxirribosa/química , Compuestos Férricos , Isoniazida/análogos & derivados , Piridoxal/análogos & derivados , Daño del ADN , Dimetilsulfóxido , Ácido Edético , Depuradores de Radicales Libres , Radical Hidroxilo , Cinética , Modelos Químicos , Plásmidos , Relación Estructura-ActividadRESUMEN
Tannic acid (TA), a plant polyphenol, has been described as having antimutagenic, anticarcinogenic and antioxidant activities. Since it is a potent chelator of iron ions, we decided to examine if the antioxidant activity of TA is related to its ability to chelate iron ions. The degradation of 2-deoxyribose induced by 6 microM Fe(II) plus 100 microM H2O2 was inhibited by TA, with an I50 value of 13 microM. Tannic acid was over three orders of magnitude more efficient in protecting against 2-deoxyribose degradation than classical *OH scavengers. The antioxidant potency of TA was inversely proportional to Fe(II) concentration, demonstrating a competition between H2O2 and AT for reaction with Fe(II). On the other hand, the efficiency of TA was nearly unchanged with increasing concentrations of the *OH detector molecule, 2-deoxyribose. These results indicate that the antioxidant activity of TA is mainly due to iron chelation rather than *OH scavenging. TA also inhibited 2-deoxyribose degradation mediated by Fe(III)-EDTA (iron = 50 microM) plus ascorbate. The protective action of TA was significantly higher with 50 microM EDTA than with 500 microM EDTA, suggesting that TA removes Fe(III) from EDTA and forms a complex with iron that cannot induce *OH formation. We also provided evidence that TA forms a stable complex with Fe(II), since excess ferrozine (14 mM) recovered 95-96% of the Fe(II) from 10 microM TA even after a 30-min exposure to 100-500 microM H2O2. Addition of Fe(III) to samples containing TA caused the formation of Fe(II)n-TA, complexes, as determined by ferrozine assays, indicating that TA is also capable of reducing Fe(III) ions. We propose that when Fe(II) is complexed to TA, it is unable to participate in Fenton reactions and mediate *OH formation. The antimutagenic and anticarcinogenic activity of TA, described elsewhere, may be explained (at least in part) by its capacity to prevent Fenton reactions.
Asunto(s)
Antioxidantes/farmacología , Compuestos Ferrosos/química , Flavonoides , Taninos Hidrolizables/farmacología , Radical Hidroxilo/antagonistas & inhibidores , Antioxidantes/química , Desoxirribosa/química , Taninos Hidrolizables/química , Fenoles/química , Fenoles/farmacología , Polímeros/química , Polímeros/farmacología , PolifenolesRESUMEN
In this report we study the effect of Fe(III) on lipid peroxidation induced by Fe(II)citrate in mitochondrial membranes, as assessed by the production of thiobarbituric acid-reactive substances and antimycin A-insensitive oxygen uptake. The presence of Fe(III) stimulates initiation of lipid peroxidation when low citrate:Fe(II) ratios are used (< or = 4:1). For a citrate:total iron ratio of 1:1 the maximal stimulation of lipid peroxidation by Fe(III) was observed when the Fe(II):Fe(III) ratio was in the range of 1:1 to 1:2. The lag phase that accompanies oxygen uptake was greatly diminished by increasing concentrations of Fe(III) when the citrate:total iron ratio was 1:1, but not when this ratio was higher. It is concluded that the increase of lipid peroxidation by Fe(III) is observed only when low citrate:Fe(II) ratios were used. Similar results were obtained using ATP as a ligand of iron. Monitoring the rate of spontaneous Fe(II) oxidation by measuring oxygen uptake in buffered medium, in the absence of mitochondria, Fe(III)-stimulated oxygen consumption was observed only when a low citrate:Fe(II) ratio was used. This result suggests that Fe(III) may facilitate the initiation and/or propagation of lipid peroxidation by increasing the rate of Fe(II)citrate-generated reactive oxygen species.
Asunto(s)
Compuestos Férricos/metabolismo , Compuestos Ferrosos/metabolismo , Peroxidación de Lípido , Lípidos de la Membrana/metabolismo , Mitocondrias/metabolismo , Animales , Ácido Cítrico , Membranas Intracelulares/metabolismo , Consumo de Oxígeno , Ratas , Ratas WistarRESUMEN
The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.
Asunto(s)
Desoxirribosa/química , Radical Hidroxilo/química , Isoniazida/análogos & derivados , Piridoxal/análogos & derivados , Óxidos N-Cíclicos , Espectroscopía de Resonancia por Spin del Electrón , Peróxido de Hidrógeno , Hierro , Isoniazida/química , Oxidación-Reducción , Piridoxal/química , Detección de SpinRESUMEN
Pyridoxal isonicotinoyl hydrazone (PIH) has previously been studied for use in iron chelation therapy in iron-overload diseases. It is an efficient in vitro antioxidant due to its Fe(III) complexing activity (Schulman, H. M., et al. Redox Report 1:373-378; 1995). Pathologies associated with iron-overload include hepatic and other cancers. Since oxidative alterations of DNA can be linked to the development of cancer, we decided to study whether PIH protects DNA against in vitro oxidative stress. We report here that pUC-18 plasmid DNA is damaged by *OH radicals generated from Fe(II) plus H2O2 or from Fe(II) plus hypoxanthine/xanthine oxidase. The DNA damage was quantified by determining the diminution of supercoiled DNA forms after oxidative attack using agar gel electrophoresis. Micromolar amounts of PIH (20-30 microM) were able to half-protect DNA from iron (1-7.5 microM)-mediated *OH formation. The antioxidant capacity of PIH was significantly higher than that of some of its analogs and desferrioxamine. PIH and some of its analogues could also inhibit the oxidative degradation of 2-deoxyribose caused by Fenton reagents. Since we observed that PIH enhances the Fe(II) autoxidation rate, measured by the ferrozine technique, PIH may limit *OH formation and consequently DNA damage by decreasing the amount of Fe(II) available to catalyze Fenton reactions.
Asunto(s)
Antioxidantes , Daño del ADN/efectos de los fármacos , Radical Hidroxilo/farmacología , Quelantes del Hierro/farmacología , Isoniazida/análogos & derivados , Plásmidos/genética , Piridoxal/análogos & derivados , Compuestos Ferrosos/química , Radical Hidroxilo/metabolismo , Hipoxantina/farmacología , Isoniazida/farmacología , Oxidación-Reducción , Piridoxal/farmacología , Xantina Oxidasa/farmacologíaRESUMEN
The roles of enzymatic antioxidant defenses in the natural tolerance of environmental stresses that impose changes in oxygen availability and oxygen consumption on animals is discussed with a particular focus on the biochemistry of estivation and metabolic depression in pulmonate land snails. Despite reduced oxygen consumption and PO2 during estivation, which should also mean reduced production of oxyradicals, the activities of antioxidant enzymes, such as superoxide dismutase and catalase, increased in 30 day-estivating snails. This appears to be an adaptation that allows the snails to deal with oxidative stress that takes place during arousal when PO2 and oxygen consumption rise rapidly. Indeed, oxidative stress was indicated by increased levels of lipid peroxidation damage products accumulating in hepatopancreas within minutes after arousal was initiated. The various metabolic sites responsible for free radical generation during arousal are still unknown but it seems unlikely that the enzyme xanthine oxidase plays any substantial role in this despite being implicated in oxidative stress in mammalian models of ischemia/reperfusion. We propose that the activation of antioxidant defenses in the organs of Otala lactea during estivation is a preparative mechanism against oxidative stress during arousal. Increased activities of antioxidant enzymes have also observed under other stress situations in which the actual production of oxyradicals should decrease. For example, antioxidant defenses are enhanced during anoxia exposure in garter snakes Thamnophis sirtalis parietalis (10 h at 5 degrees C) and leopard frogs Rana pipiens (30 h at 5 degrees C) and during freezing exposure (an ischemic condition due to plasma freezing) in T. sirtalis parietalis and wood frogs Rana sylvatica. It seems that enhancement of antioxidant enzymes during either anoxia or freezing is used as a preparatory mechanism to deal with a physiological oxidative stress that occurs rapidly within the early minutes of recovery during reoxygenation or thawing. Thus, a wide range of stress tolerant animals display coordinated changes in antioxidant defenses that allow them to deal with oxidative stress that occurs as part of natural cycles of stress/recovery that alter oxygen levels in tissues. The molecular mechanisms that trigger and regulate changes in antioxidant enzyme activities in these species are still unknown but could prove to have key relevance for the development of new intervention strategies in the treatment of cardiovascular ischemia/reperfusion injuries in humans.
Asunto(s)
Antioxidantes/metabolismo , Caracoles/metabolismo , Animales , Estivación , Humanos , Peroxidación de Lípido , Modelos Biológicos , Estrés Oxidativo , Consumo de Oxígeno , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/metabolismo , Vertebrados , Xantina Oxidasa/metabolismoRESUMEN
Many anurans have excellent dehydration tolerance that allows endurance of the loss of up to 50-60% of total body water. One of the effects of severe dehydration is circulatory impairment due the reduced volume and increased viscosity of blood, which leads to organ hypoxia. The rehydration situation, therefore, involves a reoxygenation of tissues that may include elements of oxidative stress that resemble the injury in post-ischemic reperfusion of mammalian organs. The role of endogenous defenses against oxygen radicals in the tolerance of severe dehydration by leopard frogs, Rana pipiens, was investigated by monitoring the activities of antioxidant enzymes and glutathione levels (reduced GSH and oxidized GSSG) in leg muscle and liver of control, 50%-dehydrated, and fully rehydrated frogs. The maximal activities of muscle catalase and liver glutathione peroxidase, measured per mg soluble protein, increased significantly by 52 and 74%, respectively, after dehydration whereas muscle superoxide dismutase and glutathione reductase activities responded oppositely, decreasing by 32 and 35%, respectively. Enzyme activities returned to control levels after full rehydration. Hepatic GSH and GSSG increased early in the rehydration process (30% recovery of total body water), but returned to control levels after full recovery. A similar trend was observed for liver GSSG. The elevation of antioxidant defenses against peroxides during dehydration could provide protection against post-hypoxic oxyradical stress during rehydration. Indeed, analysis of one product of lipid peroxidation, thiobarbituric acid reactive substances, in frog tissues gave no indication of oxidative stress during the dehydration/rehydration cycle.
Asunto(s)
Antioxidantes/metabolismo , Deshidratación/metabolismo , Glutatión/fisiología , Rana pipiens/fisiología , Animales , Fluidoterapia , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Glutatión Transferasa/metabolismo , Hipoxia/metabolismo , Peroxidación de Lípido , Hígado/química , Hígado/metabolismo , Masculino , Músculos/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Phospho(enol)pyruvate (PEP) undergoes transphosphorylation to form pyrophosphate (PPi) and adenosine 5'-diphosphate (5'-ADP) with high yields in the presence of an adsorbent surface of calcium phosphate (Pi.Ca), which is considered to be an ancient mineral with catalytic properties. PPi formation is a result of the phosphorolytic cleavage of the enol phosphate group of PEP by precipitated Pi. The synthesis of PPi is dependent on the amount of the solid matrix; it increases with the amount of adsorbed PEP and upon addition of dimethyl sulfoxide (Me2SO), a molecule with high dipolar moment. Although it is saturated with PEP at neutral pH, the phosphorylating Pi.Ca surface becomes effective only in alkaline conditions. In a parallel reaction, PEP phosphorylates 5'-AMP to 5'-ADP with a yield that is sevenfold higher in the presence of the Pi.Ca surface than in its absence, indicating that the solid matrix promotes interaction between adsorbed molecules with a high potential for phosphoryl transfer. In contrast to phosphorolysis, this latter reaction is stimulated by Me2SO only in homogeneous solution. It is concluded that phosphate minerals may have coadjuvated in reactions involving different phosphorylated compounds and that molecules with high dipolar moment may have acted in mildly alkaline, primitive aqueous environments to modulate phosphoryl transfer reactions catalyzed by phosphate minerals.
Asunto(s)
Adenosina Difosfato/síntesis química , Fosfatos de Calcio/química , Dimetilsulfóxido/farmacología , Difosfatos/síntesis química , Fosfoenolpiruvato/química , Adenosina Monofosfato/química , Adsorción , Catálisis , Concentración de Iones de Hidrógeno , Fosforilación , SolubilidadRESUMEN
Trifluoperazine (TFP) (35 microM) prevents mitochondrial transmembrane potential (delta psi) collapse and swelling induced by 10 microM Ca2+ plus oxyradicals generated from delta-aminolevulinic acid autoxidation. In contrast with EGTA, TFP cannot restore the totally collapsed delta psi. So, TFP might not remove Ca2+ from its 'harmful site', but could impair the ROS-driven cross-linking between membrane-SH proteins. Our data are correlated with the protective uses of TFP against oxidative processes promoted by oxyradicals plus Ca2+.
Asunto(s)
Calcio/fisiología , Mitocondrias Hepáticas/efectos de los fármacos , Especies Reactivas de Oxígeno/fisiología , Trifluoperazina/farmacología , Ácido Aminolevulínico/metabolismo , Animales , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias Hepáticas/patología , Mitocondrias Hepáticas/ultraestructura , Oxidación-Reducción , Permeabilidad/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
It is well established that several iron complexes can induce oxidative damage in hepatic mitochondrial membranes by catalyzing the formation of OH radicals and/or by promoting lipid peroxidation. This is a relevant process for the molecular basis of iron overload diseases. The present work demonstrates that Fe(II)ATP complexes (5-50 microM) promote an oxygen consumption burst in a suspension of isolated rat liver mitochondria (either in the absence or presence of Antimycin A), caused mainly by lipid peroxidation. Fe(II)ATP alone induced small levels of oxygen uptake but no burst. The time course of Fe(II)ATP oxidation to Fe(II)ATP in the extramitochondrial media also reveals a simultaneous 'burst phase'. The iron chelator Desferal (DFO) or the chain-break antioxidant butylated hydroxytoluene (BHT) fully prevented both lipid peroxidation (quantified as oxygen uptake burst) and mitochondrial swelling. DFO and BHT were capable of stopping the ongoing process of peroxidation at any point of their addition to the mitochondrial suspension. Conversely, DFO and BHT only halted the Fe(II)ATP-induced mitochondrial swelling at the onset of the process. Fe(II)ATP could also cause the collapse of mitochondrial potential, which was protected by BHT if added at the onset of the damaging process. These results, as well as correlation studies between peroxidation and mitochondrial swelling, suggest that a two phase process is occurring during Fe(II)ATP-induced mitochondrial damage: one dependent and another independent of lipid peroxidation. The involvement of lipid peroxidation in the overall process of mitochondrial membrane injury is discussed.
Asunto(s)
Adenosina Trifosfato/farmacología , Compuestos Ferrosos/farmacología , Peroxidación de Lípido , Mitocondrias Hepáticas/efectos de los fármacos , Animales , Hidroxitolueno Butilado/farmacología , Catalasa/metabolismo , Deferoxamina/farmacología , Compuestos Ferrosos/metabolismo , Técnicas In Vitro , Membranas Intracelulares/efectos de los fármacos , Potenciales de la Membrana , Dilatación Mitocondrial , Modelos Químicos , Estrés Oxidativo , Consumo de Oxígeno , Permeabilidad/efectos de los fármacos , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismo , Factores de TiempoRESUMEN
Reactive oxygen species (ROS) generated by metal-catalyzed 5-aminolevulinic acid (ALA) aerobic oxidation have been shown to damage the inner membrane of isolated rat liver mitochondria by a Ca(2+)-dependent mechanism. The present work describes experiments indicating that this damage can be prevented, but not completely reversed by the additions of catalase, ADP, cyclosporin A and dithiothreitol, as judged by the extent of delta psi regeneration by the injured mitochondria. In contrast, the addition of EGTA, which removes free Ca2+ and, possibly, Fe2+ present both in the intra- and extramitochondrial compartments, causes a prompt and complete regeneration of delta psi, even after long periods of mitochondrial incubations in the presence of ALA. This reversibility suggests that protein alterations such as protein thiol cross-linkings, evidenced by SDS-polyacrylamide gel electrophoresis, are the main cause of increased membrane permeability promoted by ALA oxidation. The inhibition of protein aggregation and fast regeneration of delta psi promoted by EGTA suggest that the binding of Ca2+ to some membrane proteins plays a crucial role in the mechanism of both protein polymerization (pore assembly) and pore opening. The implication of these results with the molecular pathology of acute intermittent porphyria is also discussed.
Asunto(s)
Ácido Aminolevulínico/farmacología , Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Compuestos de Sulfhidrilo/metabolismo , Animales , Membranas Intracelulares/metabolismo , Masculino , Potenciales de la Membrana , Mitocondrias Hepáticas/metabolismo , NADP/metabolismo , Oxidación-Reducción , Permeabilidad/efectos de los fármacos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/farmacologíaRESUMEN
Isolated rat liver mitochondria exposed to Fe(II)citrate undergo lipid peroxidation and alterations in membrane proteins. These processes were associated with irreversible decrease in membrane potential and mitochondrial swelling. Lipid peroxidation was evidenced by the production of thiobarbituric acid-reactive substances and also by the reaction of these products with membrane proteins, through the formation of Schiff bases. Alterations in membrane proteins were also characterized by the loss of specific proteins that could be recovered from the mitochondrial supernatant as shown by SDS-polyacrylamide gel electrophoresis. The degree of both lipid peroxidation and alterations in membrane proteins were diminished by EGTA, ruthenium red, or dibucaine. This strongly indicates that Ca2+ potentiates the oxidative damage of mitochondria exposed to Fe(II)citrate.
Asunto(s)
Calcio/farmacología , Compuestos Ferrosos/toxicidad , Peroxidación de Lípido/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Animales , Calcio/metabolismo , Ácido Cítrico , Sinergismo Farmacológico , Ácido Egtácico/farmacología , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Hierro/farmacología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/aislamiento & purificación , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/ultraestructura , Dilatación Mitocondrial/efectos de los fármacos , Compuestos Onio/farmacología , Compuestos Organofosforados/farmacología , Ratas , Ratas Wistar , Rojo de Rutenio/farmacología , Factores de TiempoRESUMEN
Swelling of isolated rat liver mitochondria is shown to be induced by metal-catalyzed 5-aminolevulinic acid (ALA) aerobic oxidation, a putative endogenous source of reactive oxygen species (ROS), at concentrations as low as 50-100 microM. In this concentration range, ALA is estimated to occur in the liver of acute intermittent porphyria patients. Removal of Ca2+ (10 microM) from the suspension of isolated rat liver mitochondria by added EGTA abolishes both the ALA-induced transmembrane-potential collapse and mitochondrial swelling. Prevention of the ALA-induced swelling by addition of ruthenium red prior to mitochondrial energization by succinate demonstrates the deleterious involvement of internal Ca2+. Addition of MgCl2 at concentrations higher than 2.5 mM, prevents the ALA-induced mitochondrial swelling, transmembrane potential collapse and Ca2+ efflux. This indicates that Mg2+ protects against the mitochondrial damage promoted by ALA-generated ROS. The ALA-induced mitochondrial damage might be a key event in the liver mitochondrial damage of acute intermittent porphyria patients reported elsewhere.
Asunto(s)
Ácido Aminolevulínico/farmacología , Calcio/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Ácido Aminolevulínico/antagonistas & inhibidores , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Ácido Egtácico , Magnesio/farmacología , Masculino , Mitocondrias Hepáticas/metabolismo , Oxidación-Reducción , Porfirias Hepáticas/metabolismo , Ratas , Ratas WistarRESUMEN
The enzyme-like kinetic properties of precipitated magnesium phosphate as a catalyst for formation of pyrophosphate (PPi) from phospho(enol)pyruvate (PEP) are described. This synthesis occurs at a low temperature (37 degrees C) and represents a model that may help us understand the relevance to chemical evolution of minerals as ancient catalysts whose functions could have been taken over by contemporary enzymes. An insoluble Pi.Mg matrix was formed in a medium with 80% of the water replaced by dimethyl sulfoxide as a way of simulating conditions in a drying pond. Phospho(enol)pyruvate adsorbs onto the Pi.Mg surface according to a Langmuir isotherm, and the PEP concentration dependence of PPi formation follows a Michaelian-like function. A yield of 33% for transformation of the initially adsorbed PEP into PPi was attained after 4 days of incubation with equimolecular concentrations of Pi, MgCl2, and PEP. The magnesium concentration dependence for Pi and Mg precipitation, for adsorption of PEP onto solid Pi.Mg, and for PPi formation showed complex cooperative behavior. These results taken as a whole lead to the conclusion that the Pi.Mg surface not only provides a reactant for PPi formation but also catalyzes the reaction.
Asunto(s)
Difosfatos/síntesis química , Compuestos de Magnesio , Fosfoenolpiruvato/química , Cinética , Magnesio/química , Fosfatos/química , TemperaturaRESUMEN
1. The enolamine form of 5-aminolaevulinic acid (ALA), a haem precursor that accumulates in lead poisoning and in acute intermittent porphyria (AIP), undergoes fast autoxidation at slightly alkaline pH with concomitant generation of reactive oxygen species. 2. The transmembrane potential, Ca2+ ion fluxes and state-4 respiratory rate, of isolated rat liver mitochondria are severely affected by mM addition of ALA; the toxic role of ALA-produced oxygen radicals was demonstrated by use of appropriate scavengers. 3. Induction of superoxide dismutase biosynthesis in lead-exposed workers, in AIP carriers and in ALA-treated rats, is viewed as a protective response against oxygen radical toxicity. 4. 5-Aminolaevulinic acid-generated oxygen radicals, together with Pb-stimulated Fe-dependent lipid peroxidation, might be involved in the aetiology of the neuropsychiatric manifestations of both plumbism and acute intermittent porphyria.
Asunto(s)
Radicales Libres , Intoxicación por Plomo/metabolismo , Animales , HumanosRESUMEN
delta-Aminolevulinic acid is a heme precursor accumulated in acute intermittent porphyria and lead-poisoning, which supposedly triggers the typical clinical expression associated with these diseases. Considering that: (i) erythrocyte anti-oxidant enzymes are abnormally high in patients with both disorders and (ii) delta-aminolevulinic acid autoxidation generates reactive oxygen species, a possible contribution of reactive oxygen species in the pathophysiology of these disorders is explored here. Evidence is provided that delta-aminolevulinic acid (2-15 mM) induces damage to isolated rat liver mitochondria. Addition of delta-aminolevulinic acid disrupts the mitochondrial membrane potential, promotes Ca2+ release from the intramitochondrial matrix and releases the state-4 respiration, thus enhancing the permeability of the membrane to H+. The lesion was abolished by catalase, superoxide dismutase (both enzymes inhibit delta-aminolevulinic acid autoxidation) and ortho-phenanthroline, but not by mannitol; added H2O2 induces damage poorly. These results suggest the involvement of deleterious reactive oxygen species formed at particular mitochondrial sites from transition metal ions and delta-aminolevulinic acid-generated peroxide and/or superoxide species. These observations might be compatible with previous work showing hepatic mitochondrial damage in liver biopsy samples of acute intermittent porphyria patients.
Asunto(s)
Ácido Aminolevulínico/farmacología , Intoxicación por Plomo/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Oxígeno/metabolismo , Porfirias/metabolismo , Animales , Calcio/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Endogámicas , Superóxido Dismutasa/metabolismoRESUMEN
A previous communication (Fagian, M. M., Pereira da Silva, L. and Vercesi, A. E. (1986) Biochim. Biophys. Acta 852, 262-268) indicated that intramitochondrial calcium inhibits oxidative phosphorylation by decreasing the availability of adenine nucleotides to both the ADP/ATP translocase and the F0F1-ATP synthase complex. In this work we analyzed the interactions of calcium-nucleotide and magnesium-nucleotide complexes with the ATP synthase during catalysis of ATP in equilibrium with [32P]Pi exchange and net synthesis of ATP by submitochondrial particles. Concerning the ATP in equilibrium with [32P]Pi exchange reaction, calcium was ineffective as divalent cation when assayed alone. Furthermore, the addition of calcium increased the magnesium concentration required for half-maximal activation of the exchange, without changing Vmax. With respect to net ATP synthesis, the inhibition by calcium was shown to be due to formation of the CaADP- complex, which competes with MgADP- for the active site of the F0F1-ATP synthase. Moreover, ATP hydrolysis was competitively inhibited by CaATP2-, showing that calcium is able to interact with the enzyme in both forward and backward reactions in the same manner. That high calcium concentrations are required for significant inhibition of ATP synthesis indicates that this inhibition is relevant under conditions in which cytosolic calcium concentrations rise to pathological levels. Therefore, this mechanism may be responsible, in part, for the decrease in cellular ATP content that has been observed to occur when calcium accumulates in the cytosol.