RESUMEN
BACKGROUND AND PURPOSE: The clinical utility of anthracycline antineoplastic drugs is limited by the risk of cardiotoxicity, which has been traditionally attributed to iron-mediated production of reactive oxygen species (ROS). EXPERIMENTAL APPROACH: The aims of this study were to examine the strongly lipophilic iron chelator, salicylaldehyde isonicotinoyl hydrazone (SIH), for its ability to protect rat isolated cardiomyocytes against the toxicity of daunorubicin (DAU) and to investigate the effects of SIH on DAU-induced inhibition of proliferation in a leukaemic cell line. Cell toxicity was measured by release of lactate dehydrogenase and staining with Hoechst 33342 or propidium iodide and lipid peroxidation by malonaldehyde formation. KEY RESULTS: SIH fully protected cardiomyocytes against model oxidative injury induced by hydrogen peroxide exposure. SIH also significantly but only partially and with no apparent dose-dependency, reduced DAU-induced cardiomyocyte death. However, the observed protection was not accompanied by decreased lipid peroxidation. In the HL-60 acute promyelocytic leukaemia cell line, SIH did not blunt the antiproliferative efficacy of DAU. Instead, at concentrations that reduced DAU toxicity to cardiomyocytes, SIH enhanced the tumoricidal action of DAU. CONCLUSIONS AND IMPLICATIONS: This study demonstrates that iron is most likely involved in anthracycline cardiotoxicity and that iron chelation has protective potential, but apparently through mechanism(s) other than by inhibition of ROS-induced injury. In addition to cardioprotection, iron chelation may have considerable potential to improve the therapeutic action of anthracyclines by enhancing their anticancer efficiency and this potential warrants further investigation.
Asunto(s)
Aldehídos/farmacología , Antibióticos Antineoplásicos/toxicidad , Daunorrubicina/toxicidad , Hidrazonas/farmacología , Quelantes del Hierro/farmacología , Leucemia Promielocítica Aguda/patología , Miocitos Cardíacos/efectos de los fármacos , Animales , Animales Recién Nacidos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citoprotección , Relación Dosis-Respuesta a Droga , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The aim of this study was to analyze the ECG time intervals in the course of the development of chronic anthracycline cardiomyopathy in rabbits. Furthermore, this approach was employed to study the effects of a model cardioprotective drug (dexrazoxane) and two novel iron chelating compounds--salicylaldehyde isonicotinoyl hydrazone (SIH) and pyridoxal 2-chlorobenzoyl hydrazone (o-108). Repeated daunorubicin administration induced a significant and progressive prolongation of the QRS complex commencing with the eighth week of administration. At the end of the study, we identified a significant correlation between QRS duration and the contractility index dP/dt(max) (r = -0.81; P<0.001) as well as with the plasma concentrations of cardiac troponin T (r = 0.78; P<0.001). In contrast, no alterations in ECG time intervals were revealed in the groups co-treated with either dexrazoxane or both novel cardioprotective drugs (SIH, o-108). Hence, in this study, the QRS duration is for the first time shown as a parameter suitable for the non-invasive evaluation of the anthracycline cardiotoxicity and cardioprotective effects of both well established and investigated drugs. Moreover, our results strongly suggest that novel iron chelators (SIH and o-108) merit further study as promising cardioprotective drugs against anthracycline cardiotoxicity.
Asunto(s)
Cardiomiopatías/prevención & control , Cardiotónicos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Electrocardiografía , Sistema de Conducción Cardíaco/efectos de los fármacos , Quelantes del Hierro/farmacología , Aldehídos/farmacología , Animales , Cardiomiopatías/sangre , Cardiomiopatías/inducido químicamente , Cardiomiopatías/fisiopatología , Cardiotónicos/uso terapéutico , Enfermedad Crónica , Daunorrubicina , Modelos Animales de Enfermedad , Sistema de Conducción Cardíaco/fisiopatología , Hidrazonas/farmacología , Quelantes del Hierro/uso terapéutico , Masculino , Contracción Miocárdica/efectos de los fármacos , Piridoxal/análogos & derivados , Piridoxal/farmacología , Conejos , Razoxano/farmacología , Factores de Tiempo , Troponina T/sangreRESUMEN
Recently, pyridoxal 2-chlorobenzoyl hydrazone (o-108) has been identified as an effective iron chelator [Link et al., Blood 2003; 101: 4172-79]. Since chronic treatment would be necessary in its potential indications, in the present study, the safety and tolerability of this agent after repeated administration was determined. Three doses of o-108 (25, 50, 100 mg/kg, in 10% Cremophor EL) were administered intraperitoneally, once weekly, for 10 weeks to three groups (n=5 each) of Chinchilla male rabbits. The effects on biochemical, haematological and cardiovascular parameters were examined during the experiment; histopathological examination was performed at the end of the experiment. Results were compared with control (saline 2 mL/kg, n=11) and vehicle groups (10% Cremophor EL, 2 mL/kg, n=12). No premature deaths occurred; the well-being of animals was evidenced by their body weight gain, although lower gain was observed with the highest dose (100 mg/kg). Significant elevations of cardiac troponin T plasma concentrations were observed with the highest dose of o-108, but no abnormalities were found in the cardiovascular function and only minor and inconsistent changes in haematological and biochemical parameters were observed. Histopathological examinations of selected organs revealed only weak and reversible changes through all studied groups. Thus, the data from this study suggest that o-108 remains a promising drug from the standpoint of the possibility of its repeated administration and warrants further investigation.
Asunto(s)
Hidrazonas/toxicidad , Quelantes del Hierro/toxicidad , Piridoxal/análogos & derivados , Animales , Recuento de Células Sanguíneas , Peso Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Enzimas/sangre , Hidrazonas/administración & dosificación , Hidrazonas/farmacocinética , Quelantes del Hierro/administración & dosificación , Masculino , Microscopía Electrónica de Rastreo , Piridoxal/administración & dosificación , Piridoxal/farmacocinética , Piridoxal/toxicidad , Conejos , Factores de Tiempo , Distribución Tisular , Troponina T/sangreRESUMEN
With the aim of identifying an iron (Fe) chelator which is effective at mobilizing intracellular Fe, two novel ligands were synthesized and tested. Hydroxyquinoline is known to possess a high affinity for Fe and was thus chosen as the Fe binding motif for the hexadentate chelators, C1 (2,2'-[ethane-1,2-diylbis(iminomethylene)]diquinolin-8-ol) and C2 (2,2'-[cyclohexane-1,2-diylbis(iminomethylene)]diquinolin-8-ol). Both chelators are lipophilic, with Fe3+ complexes slightly more hydrophilic than the free ligands. C1 and C2 were equally toxic to K562 cells, and partial protection was afforded by supplementing the culture medium with human holotransferrin, suggesting that some of the toxicity of the ligands is due to cellular Fe depletion. Micromolar concentrations of both ligands effectively mobilized 59Fe from reticulocytes and K562 cells. In reticulocytes, 50 microM C1 caused the release of 60% of the cells' initial 59Fe uptake after a 4h incubation. Under the same conditions, C2 revealed a release of 50% of the 59Fe. Overall, both ligands merit in vivo study for oral activity. Their effectiveness at low concentrations makes them candidates for therapeutic use.
Asunto(s)
Quelantes/farmacología , Ciclohexilaminas/farmacología , Etilenodiaminas/farmacología , Hidroxiquinolinas/farmacología , Hierro/metabolismo , Quelantes/toxicidad , Ciclohexilaminas/toxicidad , Etilenodiaminas/toxicidad , Humanos , Hidroxiquinolinas/toxicidad , Células K562 , Reticulocitos/efectos de los fármacos , Reticulocitos/metabolismo , Espectrofotometría/métodosRESUMEN
Risk of cardiotoxicity is the most serious drawback of the clinical usefulness of anthracycline antineoplastic antibiotics, which however, remain among the most powerful and widely employed anticancer drugs. In this study we have used daunorubicin-induced cardiomyopathy in rabbits as a model to investigate possible cardioprotective effects of pyridoxal isonicotinoyl hydrazone (PIH)-a principal representative of a novel group of aroylhydrazone iron chelators. Three groups of animals were used: a control group (n=11; i.v. saline), daunorubicin-treated animals (n=11; 3mg/kg, i.v.), and animals pretreated with PIH (n=9, 25 mg/kg, i.p.) 60 min before daunorubicin administration. All substances were administered once weekly for 10 weeks. Repeated administration of daunorubicin caused premature death in four animals and induced conspicuous histopathological changes in the myocardium, progressive and significant impairment of systolic heart function (a decrease in left ventricular dP/dt(max), ejection fraction, an increase in the pre-ejection period/left ventricular ejection time index), and a gradual increase in cardiac troponin T plasma concentrations. On the contrary, all the PIH-treated animals have survived all daunorubicin applications. Furthermore, in this group, the daunorubicin-induced cardiac changes were in most functional, biochemical as well as morphological parameters less pronounced than in the group receiving daunorubicin alone. Hence, PIH and other aroylhydrazones merit further investigation as potentially protective agents against anthracycline-induced cardiotoxicity.
Asunto(s)
Cardiotónicos/farmacología , Daunorrubicina/toxicidad , Isoniazida/análogos & derivados , Isoniazida/farmacología , Miocardio/metabolismo , Piridoxal/análogos & derivados , Piridoxal/farmacología , Animales , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Masculino , Miocardio/patología , ConejosRESUMEN
Pyridoxal isonicotinoyl hydrazone (PIH) is a new tridentate Fe-chelating agent that should be very promising in many pathological states resulting from both an iron-overload and formation of free radicals. The aim of our study was to investigate the effect of PIH on the cardiovascular system focusing to the regulatory protein -- cardiac troponin T (cTnT). The study was carried out in two groups of Chinchilla male rabbits: 1) PIH (50 mg/kg dissolved in 10 % Cremophor i.p., once a week, 10 administrations, n=8) and 2) Cremophor (2 ml/kg i.p. in the same schedule, n=7). Plasma concentrations of cTnT (as a marker of myocardial damage) were measured using a commercial kit (Roche). cTnT was within the physiological range (i.e. < 0.1 microg/l) during the whole experiment in the Cremophor group. In the PIH group, the cTnT levels were not significantly increased when compared with the control group or with the initial values (except with those before the 5th administration). Furthermore, we analyzed the cytosolic and myofibrillar fraction of cTnT in the left ventricular myocardium. Using SDS-PAGE and Western blot we resolved three isoforms. The profiling of TnT did not differ significantly between the PIH-treated group and the Cremophor-treated group. Our data concerning cTnT support the opinion that the possible cardiotoxicity of PIH is very low.
Asunto(s)
Quelantes del Hierro/toxicidad , Isoniazida/análogos & derivados , Isoniazida/toxicidad , Miocardio/metabolismo , Piridoxal/análogos & derivados , Piridoxal/toxicidad , Troponina T/sangre , Animales , Fraccionamiento Celular/métodos , Citosol/metabolismo , Sobrecarga de Hierro/tratamiento farmacológico , Masculino , Miofibrillas/metabolismo , ConejosRESUMEN
Pyridoxal isonicotinoyl hydrazone (PIH) analogues are effective iron chelators in vivo and in vitro, and may be of value for the treatment of secondary iron overload. The sensitivity of Jurkat cells to Fe-chelator complexes was enhanced several-fold by the depletion of the antioxidant glutathione, indicating the role of oxidative stress in their toxicity. K562 cells loaded with eicosapentaenoic acid, a fatty acid particularly susceptible to oxidation, were also more sensitive to the toxic effects of the Fe complexes, and toxicity was proportional to lipid peroxidation. Thus Fe-chelator complexes cause oxidative stress, which may be a major component of their toxicity. As was the case for their Fe complexes, the toxicity of PIH analogues was enhanced by glutathione depletion of Jurkat cells and eicosapentaenoic acid-loading of K562 cells. Thus the toxicity of the chelators themselves is also enhanced by compromised cellular redox status. In addition, the toxicity of the chelators was diminished by culturing Jurkat cells under hypoxic conditions, which may limit the production of the reactive oxygen species that initiate oxidative stress. A significant part of the toxicity of the chelators may be due to intracellular formation of Fe-chelator complexes, which oxidatively destroy the cell.
Asunto(s)
Quelantes/toxicidad , Isoniazida/análogos & derivados , Isoniazida/toxicidad , Piridoxal/análogos & derivados , Piridoxal/toxicidad , Ácido Ascórbico , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , Humanos , Quelantes del Hierro/toxicidad , Células Jurkat , Células K562 , Cinética , Estructura Molecular , Oxidación-Reducción , Estrés Oxidativo , Relación Estructura-ActividadRESUMEN
Immature erythroid cells have an exceptionally high capacity to synthesize haem that is, at least in part, the result of the unique control of iron metabolism in these cells. In erythroid cells the vast majority of Fe released from endosomes must cross both the outer and the inner mitochondrial membranes to reach ferrochelatase, which inserts Fe into protoporphyrin IX. Based on the fact that Fe is specifically targeted into erythroid mitochondria, we have proposed that a transient mitochondria-endosome interaction is involved in Fe transfer to ferrochelatase [Ponka (1997) Blood 89, 1-25]. In this study, we examined whether the inhibition of endosome mobility within erythroid cells would decrease the rate of (59)Fe incorporation into haem. We found that, in reticulocytes, the myosin light-chain kinase inhibitor, wortmannin, and the calmodulin antagonist, W-7, caused significant inhibition of (59)Fe incorporation from (59)Fe-transferrin-labelled endosomes into haem. These results, together with confocal microscopy studies using transferrin and mitochondria labelled by distinct fluorescent markers, suggest that, in erythroid cells, endosome mobility, and perhaps their contact with mitochondria, plays an important role in a highly efficient utilization of iron for haem synthesis.
Asunto(s)
Células Precursoras Eritroides/metabolismo , Hierro/sangre , Mitocondrias/metabolismo , Reticulocitos/metabolismo , Animales , Endocitosis , Hemo/biosíntesis , Mamíferos , Modelos BiológicosRESUMEN
Divalent metal transporter 1 (DMT1) is the major transferrin-independent iron uptake system at the apical pole of intestinal cells, but it may also transport iron across the membrane of acidified endosomes in peripheral tissues. Iron transport and expression of the 2 isoforms of DMT1 was studied in erythroid cells that consume large quantities of iron for biosynthesis of hemoglobin. In mk/mk mice that express a loss-of-function mutant variant of DMT1, reticulocytes have a decreased cellular iron uptake and iron incorporation into heme. Interestingly, iron release from transferrin inside the endosome is normal in mk/mk reticulocytes, suggesting a subsequent defect in Fe(++) transport across the endosomal membrane. Studies by immunoblotting using membrane fractions from peripheral blood or spleen from normal mice where reticulocytosis was induced by erythropoietin (EPO) or phenylhydrazine (PHZ) treatment suggest that DMT1 is coexpressed with transferrin receptor (TfR) in erythroid cells. Coexpression of DMT1 and TfR in reticulocytes was also detected by double immunofluorescence and confocal microscopy. Experiments with isoform-specific anti-DMT1 antiserum strongly suggest that it is the non-iron-response element containing isoform II of DMT1 that is predominantly expressed by the erythroid cells. As opposed to wild-type reticulocytes, mk/mk reticulocytes express little if any DMT1, despite robust expression of TfR, suggesting a possible effect of the mutation on stability and targeting of DMT1 isoform II in these cells. Together, these results provide further evidence that DMT1 plays a central role in iron acquisition via the transferrin cycle in erythroid cells.
Asunto(s)
Anemia/sangre , Proteínas de Transporte de Catión/sangre , Eritrocitos/química , Proteínas de Unión a Hierro , Animales , Transporte Biológico/genética , Células CHO , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/fisiología , Cricetinae , Endosomas/química , Endosomas/metabolismo , Membrana Eritrocítica/metabolismo , Eritrocitos/ultraestructura , Células Precursoras Eritroides/química , Células Precursoras Eritroides/ultraestructura , Eritropoyetina/farmacología , Técnica del Anticuerpo Fluorescente , Hemo/metabolismo , Hemoglobinas/biosíntesis , Homocigoto , Hierro/sangre , Leucemia Eritroblástica Aguda , Ratones , Ratones Mutantes , Microscopía Confocal , Mutación , Fenilhidrazinas/farmacología , Isoformas de Proteínas/genética , Recuento de Reticulocitos , Reticulocitos/efectos de los fármacos , Reticulocitos/metabolismo , Bazo/efectos de los fármacos , Bazo/metabolismo , Transferrina/metabolismo , Células Tumorales CultivadasRESUMEN
Friedreich's ataxia (FA) is a crippling neurodegenerative disease that is due to iron (Fe) overload within the mitochondrion. One therapeutic intervention may be the development of a chelator that could remove mitochondrial Fe. We have implemented the only well characterized model of mammalian mitochondrial Fe overload to examine the Fe chelation efficacy of novel chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) class. In this model we utilize reticulocytes treated with the haem synthesis inhibitor succinylacetone which results in mitochondrial Fe-loading. Our experiments demonstrate that in contrast to desferrioxamine, several of the PCIH analogues show very high activity at mobilizing (59)Fe from (59)Fe-loaded reticulocytes. Further studies on these ligands in animals are clearly warranted considering their potential to treat FA.
Asunto(s)
Ataxia de Friedreich/tratamiento farmacológico , Quelantes del Hierro/síntesis química , Isoniazida/análogos & derivados , Isoniazida/síntesis química , Piridoxal/análogos & derivados , Piridoxal/síntesis química , Animales , Diseño de Fármacos , Ataxia de Friedreich/inducido químicamente , Ataxia de Friedreich/metabolismo , Hierro/metabolismo , Hierro/farmacología , Quelantes del Hierro/farmacología , Radioisótopos de Hierro , Isoniazida/farmacología , Ligandos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estructura Molecular , Fenilhidrazinas , Piridoxal/farmacología , Reticulocitos/efectos de los fármacos , Reticulocitos/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
During acute graft-versus-host disease (GVHD) the activation of macrophages (Mphi) is mediated by 2 signals, interferon (IFN)-gamma and bacteria-derived lipopolysaccharide (LPS). A cascade of inflammatory responses that includes the release of mediators of tissue injury follows Mphi activation. Among the tissues characteristically targeted during acute GVHD are epithelial tissues of the skin and gastrointestinal tract that normally undergo continuous proliferation and are therefore sensitive to cytostatic processes. We have investigated whether Mphi can mediate cytostatic mechanisms capable of interrupting cell proliferation during acute GVHD. GVHD was induced in nonirradiated C57BL/6XAF(1) (B6AF(1)) mice by the injection of 60 x 10(6) (acute GVHD) or 30 x 10(6) (nonlethal GVHD) C57BL/6 (B6) lymphoid cells. Mphi from animals undergoing acute GVHD could be triggered by normally insignificant quantities of LPS to mediate a cytostatic effect on target cells, resulting in the complete shutdown of cellular proliferation. The same amounts of LPS had no effect on Mphi from normal or syngeneically transplanted animals. Mphi mediated the release of significant quantities of intracellular iron from target cells undergoing cytostasis. Reversal of cytostasis occurred following inhibition of nitric oxide (NO) production by N(G)-monomethyl-L-arginine (NMMA). Production of NO by LPS-triggered Mphi reflected the severity of GVHD. NO release increased significantly during acute GVHD but was only transiently increased during nonlethal GVHD. The results provide evidence that, as a result of activation during acute GVHD, Mphi produce NO and induce the release of iron from target cells, resulting in a potent cytostatic effect that inhibits cellular proliferation. (Blood. 2000;96:1836-1843)
Asunto(s)
Enfermedad Injerto contra Huésped/patología , Macrófagos/citología , Enfermedad Aguda , Animales , División Celular/efectos de los fármacos , Línea Celular , Técnicas de Cocultivo , Citotoxicidad Inmunológica , Relación Dosis-Respuesta a Droga , Femenino , Enfermedad Injerto contra Huésped/metabolismo , Humanos , Interferón gamma/farmacología , Hierro/metabolismo , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Óxido Nítrico/metabolismo , Índice de Severidad de la Enfermedad , Factores de Tiempo , Células Tumorales Cultivadas , omega-N-Metilarginina/farmacologíaRESUMEN
OBJECTIVE: This study was designed to investigate the cardioprotective effect of the novel lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) against the oxidative stress exerted by H(2)O(2) through the production of OH radical via the Fenton reaction and to compare them with those of the hydrophilic iron chelator deferoxamine (DFO) and the Na(+)/H(+) exchange inhibitor methylisobutyl amiloride (MIA). METHODS: We used long-term cultures of spontaneously beating adult guinea-pig ventricular cardiomyocytes developed and characterized previously in our laboratory. We assessed their contractile activity by video-recording as well as the underlying Ca(i)(2+) transients by Fura 2 fluorescence. In some experiments we also recorded these functional parameters, plus the electrical activity (action potentials) in response to electrical stimulation via suction pipettes, in individual freshly isolated myocytes. RESULTS: Exposure of the regularly and synchronously beating cultured cardiomyocytes to 100 microM H(2)O(2) initially caused a substantial prolongation of Ca(i)(2+) transients accompanied by an irregular contractile activity, then in contractile arrest and a severalfold increase in cytosolic [Ca(2+)] that occurred, within 30 min of H(2)O(2) application. Similar effects were also observed using freshly isolated cardiomyocytes. The latter effects were first accompanied by significant prolongation of the action potential duration (APD) with superimposed early afterdepolarizations followed by a second phase with a very fast decrease in APD, contractions, as well as Ca(i)(2+) transients and a third phase of inexcitability, contractile arrest, increased cytoplasmic [Ca(2+)] and a final contracture. All these effects were irreversible in both types of preparations but they could be fully prevented by a 15-min preincubation with 200 microM SIH. Similar protective effects were observed with DFO, but in this case a much higher concentration had to be used (1 mM) and much longer (2 h) preincubation was needed. By contrast, 5 microM MIA failed to fully protect the cardiomyocytes, although a significant delay (10 min) of the effects of H(2)O(2) was observed. CONCLUSIONS: The data indicate that SIH provides a very powerful and very fast protection against the oxidative stress exerted by H(2)O(2) presumably via the iron-mediated Fenton reaction producing hydroxyl radical (OH), whereas the protective effect of DFO is hindred by its very slow and rather limited intracellular entry, and the protection that MIA exerts via the inhibition of Na(+)/H(+) exchange against H(2)O(2) much less effective.
Asunto(s)
Aldehídos/farmacología , Hidrazonas/farmacología , Quelantes del Hierro/farmacología , Miocardio/metabolismo , Estrés Oxidativo/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Canales de Calcio/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Deferoxamina/farmacología , Cobayas , Peróxido de Hidrógeno/farmacología , Técnicas de Placa-Clamp , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidoresRESUMEN
Hemoglobin synthesis consumes most of the iron that is taken up by cells from plasma transferrin, and this process requires very high expression of transferrin receptors (TfR) at the membranes of erythroid cells. Studies in our and other laboratories indicate that a dramatic increase in TfR levels during erythroid differentiation occurs at the transcriptional level. In this study, we investigated the transcriptional regulation of the TfR in terms of its promoter activity and DNA-protein binding in murine erythroleukemia cells. Reporter gene assays revealed that the TfR promoter activity was stimulated 6-8-fold in murine erythroleukemia cells induced to differentiate into hemoglobin-synthesizing cells by either Me(2)SO or N,N'-hexamethylene-bis-acetamide. A minimal region (-118 to +14) was required for the differentiation-induced promoter activity. Mutation of either an Ets-binding site or an activator protein-1/cyclic AMP-response element-like motif within this region, but not disruption of the adjacent GC-rich/specificity protein-1 sequence, inhibited the inducible promoter activity. Electrophoresis mobility shift assays suggest that the cyclic AMP-response element-binding proteins/activating transcription factor-like factors and Ets-like factors bind constitutively to this bipartite element. Upon induction of differentiation, a shift in the pattern of the cyclic AMP-response element-binding protein/activating transcription factor-like binding factors was observed. Our data indicate that the TfR gene promoter contains an erythroid active element that stimulates the receptor gene transcription upon induction of hemoglobin synthesis.
Asunto(s)
Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Receptores de Transferrina/genética , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción Activadores , Secuencia de Bases , Sitios de Unión , Proteínas Sanguíneas/metabolismo , Diferenciación Celular , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets , Receptores de Estrógenos/metabolismo , Elementos de Respuesta , Factores de Transcripción/metabolismo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Iron regulatory proteins (IRP-1 and IRP-2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements, which are located in the 3'-untranslated region and the 5'-untranslated region of their respective mRNAs. Cellular iron levels affect binding of IRPs to iron-responsive elements and consequently expression of TfR and ferritin. Moreover, NO(*), a redox species of nitric oxide that interacts primarily with iron, can activate IRP-1 RNA binding activity resulting in an increase in TfR mRNA levels. Recently we found that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO(+) (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA binding of IRP-2 followed by IRP-2 degradation, and these changes were associated with a decrease in TfR mRNA levels (Kim, S., and Ponka, P. (1999) J. Biol. Chem. 274, 33035-33042). In this study, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) increased IRP-1 binding activity, whereas RNA binding of IRP-2 decreased and was followed by a degradation of this protein. Moreover, the decrease of IRP-2 binding/protein levels was associated with a decrease in TfR mRNA levels in LPS/IFN-gamma-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. Furthermore, LPS/IFN-gamma-stimulated RAW 264.7 cells showed increased rates of ferritin synthesis. These results suggest that NO(+)-mediated degradation of IRP-2 plays a major role in iron metabolism during inflammation.
Asunto(s)
Interferón gamma/farmacología , Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Óxido Nítrico/farmacología , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular , Inhibidores Enzimáticos/farmacología , Compuestos Férricos/farmacología , Ferritinas/biosíntesis , Proteína 1 Reguladora de Hierro , Proteína 2 Reguladora de Hierro , Proteínas Reguladoras del Hierro , Ratones , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Unión Proteica/efectos de los fármacos , Compuestos de Amonio Cuaternario/farmacología , ARN Mensajero/metabolismo , Receptores de Transferrina/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The transferrin receptor is a membrane glycoprotein whose only clearly defined function is to mediate cellular uptake of iron from a plasma glycoprotein, transferrin. Iron uptake from transferrin involves the binding of transferrin to the transferrin receptor, internalization of transferrin within an endocytic vesicle by receptor-mediated endocytosis and the release of iron from the protein by a decrease in endosomal pH. With the exception of highly differentiated cells, transferrin receptors are probably expressed on all cells but their levels vary greatly. Transferrin receptors are highly expressed on immature erythroid cells, placental tissue, and rapidly dividing cells, both normal and malignant. In proliferating nonerythroid cells the expression of transferrin receptors is negatively regulated post-transcriptionally by intracellular iron through iron responsive elements (IREs) in the 3' untranslated region of transferrin receptor mRNA. IREs are recognized by specific cytoplasmic proteins (IRPs; iron regulatory proteins) that, in the absence of iron in the labile pool, bind to the IREs of transferrin receptor mRNA, preventing its degradation. On the other hand, the expansion of the labile iron pool leads to a rapid degradation of transferrin receptor mRNA that is not protected since IRPs are not bound to it. However, some cells and tissues with specific requirements for iron probably evolved mechanisms that can override the IRE/IRP-dependent control of transferrin receptor expression. Erythroid cells, which are the most avid consumers of iron in the organism, use a transcriptional mechanism to maintain very high transferrin receptor levels. Transcriptional regulation is also involved in the receptor expression during T and B lymphocyte activation. Macrophages are another example of a cell type that shows 'unorthodox' responses in terms of IRE/IRP paradigm since in these cells elevated iron levels increase (rather than decrease) transferrin receptor mRNA and protein levels. Erythroid cells contain the highest mass of the total organismal transferrin receptors which are released from reticulocytes during their maturation to erythrocytes. Hence, plasma contains small amounts of transferrin receptors which represent a soluble fragment of the extracellular receptor domain. Measurements of serum transferrin receptor concentrations are clinically useful since their levels correlate with the total mass of immature erythroid cells.
Asunto(s)
Receptores de Transferrina/fisiología , Animales , Regulación de la Expresión Génica , Estado de Salud , Humanos , Hipoxia/fisiopatología , Receptores de Transferrina/sangre , Receptores de Transferrina/genéticaRESUMEN
Cellular iron storage and uptake are coordinately regulated post-transcriptionally by cytoplasmic factors, iron-regulatory proteins 1 and 2 (IRP-1 and IRP-2). When iron in the intracellular transit pool is scarce, IRPs bind to iron-responsive elements (IREs) in the 5'-untranslated region of the ferritin mRNA and 3'-untranslated region of the transferrin receptor (TfR) mRNA. Such binding inhibits translation of ferritin mRNA and stabilizes the mRNA for TfR, whereas the opposite scenario develops when iron in the transit pool is plentiful. However, we (Richardson, D. R., Neumannova, V., Nagy, E., and Ponka, P. (1995) Blood 86, 3211-3219) and others reported that the binding of IRPs to IREs can also be modulated by nitric oxide (NO). In this study, we showed that a short exposure of RAW 264.7 cells (a murine macrophage cell line) to the NO(+) donor, sodium nitroprusside (SNP), caused a significant decrease in IRP-2 binding to the IREs followed by IRP-2 degradation and that these changes occurred without affecting IRP-1 binding. The SNP-mediated degradation of IRP-2 in RAW 264.7 cells could be prevented by MG-132 or lactacystin, known inhibitors of proteasome-dependent protein degradation. A SNP-mediated decrease in IRP-2 binding and levels was associated with a dramatic decrease in TfR mRNA levels and an increase in ferritin synthesis. Importantly, the proteasome inhibitor MG-132 prevented the SNP-mediated decrease in TfR mRNA levels. These observations suggest that IRP-2 can play an important role in controlling transferrin receptor expression.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Óxido Nítrico/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Transferrina/genética , Animales , Línea Celular , Cisteína Endopeptidasas/metabolismo , Deferoxamina/farmacología , Compuestos Férricos/farmacología , Hierro/metabolismo , Proteína 1 Reguladora de Hierro , Proteína 2 Reguladora de Hierro , Proteínas Reguladoras del Hierro , Proteínas Hierro-Azufre/metabolismo , Macrófagos , Ratones , Complejos Multienzimáticos/metabolismo , Óxido Nítrico/química , Nitroprusiato/farmacología , Penicilamina/análogos & derivados , Penicilamina/farmacología , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Compuestos de Amonio Cuaternario/farmacología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Tiempo , Proteína wnt2RESUMEN
Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. Heme serves as the prosthetic group of numerous hemoproteins (eg, hemoglobin, myoglobin, cytochromes, guanylate cyclase, and nitric oxide synthase) and plays an important role in controlling protein synthesis and cell differentiation. Cellular heme levels are tightly controlled; this is achieved by a fine balance between heme biosynthesis and catabolism by the enzyme heme oxygenase. On a per-cell basis, the rate of heme synthesis in the developing erythroid cells is at least 1 order of magnitude higher than in the liver, which is in turn the second most active heme producer in the organism. Differences in iron metabolism and in genes for 5-aminolevulinic acid synthase (ALA-S, the first enzyme in heme biosynthesis) are responsible for the differences in regulation and rates of heme synthesis in erythroid and nonerythroid cells. There are 2 different genes for ALA-S, one of which is expressed ubiquitously (ALA-S1), whereas the expression of the other (ALA-S2) is specific to erythroid cells. Because the 5'-untranslated region of the erythroid-specific ALA-S2 mRNA contains the iron-responsive element, a cis-acting sequence responsible for translational induction of erythroid ALA-S2 by iron, the availability of iron controls protoporphyrin IX levels in hemoglobin-synthesizing cells. In nonerythroid cells, the rate-limiting step of heme production is catalyzed by ALA-S1, whose synthesis is feedback-inhibited by heme. On the other hand, in erythroid cells, heme does not inhibit either the activity or the synthesis of ALA-S but does inhibit cellular iron acquisition from transferrin without affecting its utilization for heme synthesis. This negative feedback is likely to explain the mechanism by which the availability of transferrin iron limits heme synthesis rate. Moreover, in erythroid cells heme seems to enhance globin gene transcription, is essential for globin translation, and supplies the prosthetic group for hemoglobin assembly. Heme may also be involved in the expression of other erythroid-specific proteins. Furthermore, heme seems to play a role in regulating either transcription, translation, processing, assembly, or stability of hemoproteins in nonerythroid cells. Heme oxygenase, which catalyzes heme degradation, seems to be an important enzymatic antioxidant system, probably by providing biliverdin, which is an antioxidant agent.
Asunto(s)
Hemo/metabolismo , 5-Aminolevulinato Sintetasa/metabolismo , Animales , Monóxido de Carbono/metabolismo , Eritrocitos/metabolismo , Hemo/biosíntesis , Hemo Oxigenasa (Desciclizante)/metabolismo , Humanos , Proteínas Reguladoras del Hierro , Proteínas Hierro-Azufre/metabolismo , Óxido Nítrico/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores de Transferrina/metabolismoRESUMEN
Expression of the transferrin receptor, which mediates iron uptake from transferrin, is negatively regulated post-transcriptionally by intracellular iron through iron-responsive elements in the 3'-untranslated region of the transferrin receptor mRNA. Transcriptional mechanisms are also involved in receptor expression, but these are poorly understood. In this study we have characterized the transferrin receptor promoter region and identified a functional hypoxia response element that contains a binding site for hypoxia-inducible factor-1 (HIF-1). Exposure of K562 and HeLa cells to hypoxia for 16 h resulted in a 2- to 3-fold increase in transferrin receptor mRNA expression. A motif with multipartite organization similar to the hypoxia response element of a number of hypoxia-inducible genes such as erythropoietin was identified within a 100-base pair sequence upstream of the transcriptional start site. Mutation of a site similar to the consensus HIF-binding site (HBS) in this motif attenuated the hypoxic response by 80%. Transient co-expression of the two HIF-1 subunits (HIF-1alpha and HIF-1beta) enhanced the wild type transferrin receptor promoter activity, but that which contained a mutated HBS yielded no such response. Electrophoretic mobility shift assays revealed that HIF-1 was stimulated and bound to the transferrin receptor HBS upon hypoxic challenge. Our results indicate that the transferrin receptor is a target gene for HIF-1.
Asunto(s)
Hipoxia de la Célula/genética , Receptores de Transferrina/genética , Elementos de Respuesta , Factores de Transcripción , Secuencia de Bases , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Células HeLa , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Hierro/metabolismo , Proteínas Reguladoras del Hierro , Proteínas Hierro-Azufre/metabolismo , Células K562 , Datos de Secuencia Molecular , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas , Proteínas de Unión al ARN/metabolismoRESUMEN
Genetic studies in animal models of microcytic anemia and biochemical studies of transport have implicated the Nramp2 gene in iron transport. Nramp2 generates two alternatively spliced mRNAs that differ at their 3' untranslated region by the presence or absence of an iron-response element (IRE) and that encode two proteins with distinct carboxy termini. Antisera raised against Nramp2 fusion proteins containing either the carboxy or amino termini of Nramp2 and that can help distinguish between the two Nramp2 protein isoforms (IRE: isoform I; non-IRE: isoform II) were generated. These antibodies were used to identify the cellular and subcellular localization of Nramp2 in normal tissues and to study possible regulation by dietary iron deprivation. Immunoblotting experiments with membrane fractions from intact organs show that Nramp2 is expressed at low levels throughout the small intestine and to a higher extent in kidney. Dietary iron starvation results in a dramatic upregulation of the Nramp2 isoform I in the proximal portion of the duodenum only, whereas expression in the rest of the small intestine and in kidney remains largely unchanged in response to the lack of dietary iron. In proximal duodenum, immunostaining studies of tissue sections show that Nramp2 protein expression is abundant under iron deplete condition and limited to the villi and is absent in the crypts. In the villi, staining is limited to the columnar absorptive epithelium of the mucosa (enterocytes), with no expression in mucus-secreting goblet cells or in the lamina propria. Nramp2 expression is strongest in the apical two thirds of the villi and is very intense at the brush border of the apical pole of the enterocytes, whereas the basolateral membrane of these cells is negative for Nramp2. These results strongly suggest that Nramp2 is indeed responsible for transferrin-independent iron uptake in the duodenum. These findings are discussed in the context of overall mechanisms of iron acquisition by the body.