RESUMEN
BACKGROUND: Severe hemolytic disease of the newborn leads to the release of pro-oxidative free heme (FH). Heme oxygenase (HO) is primarily responsible for detoxifying FH. OBJECTIVE: To investigate the protective effects of HO in a model of heme overload. METHODS: For in vitro studies, NIH3T3 HO-1-luc cells were incubated with 10, 30, or 60 µM FH or methemalbumin (MHA). HO-1 promoter activity was assessed 3, 6, and 24 h after treatment. Cell survival was indexed by viability assays. For in vivo studies, 1- and 5-week-old wild-type (Wt) or HO-1-heterozygous (Het, HO-1+/-) mice were given 60 µmol FH or MHA/kg intraperitoneally. After 24 h, plasma aspartate aminotransferease (AST)/alanine transaminase (ALT) and hemopexin, liver HO activity, and lipid peroxidation (LP) were determined. RESULTS: In HO-1-luc cells, HO-1 promoter activity peaked 6 h after incubation with 30 µM FH (1.6-fold) or 60 µM MHA (2.1-fold) over baseline. Twenty-four hours after exposure to 60 µM FH, a decrease in viability of 80% was found, compared with no decrease after exposure to 60 µM MHA. In 1-week-old Wt and HO-1 Het pups given 60 µmol FH/kg, HO activity significantly increased 3.5- and 3.1-fold, respectively. No changes in LP or AST/ALT levels were observed. In adult Wt and HO-1 Het mice, HO activity increased (3.0- and 2.6-fold, respectively). LP and AST levels significantly increased 28.4- and 2.7-fold, respectively, in adult HO-1 Het mice. Hemopexin levels at baseline were higher in adults compared with newborns for both Wt and Het mice. In addition, FH induced hemopexin levels in both adults and newborns, but to a lesser degree in newborns. CONCLUSIONS: FH is highly toxic in vitro, but its toxicity is abolished when bound to albumin. Newborns appear to be protected from the pro-oxidative effects of FH, which may be mediated by heme binding and a higher absolute HO activity at baseline and after FH-mediated induction.
Asunto(s)
Eritroblastosis Fetal/enzimología , Eritrocitos/enzimología , Hemo-Oxigenasa 1/sangre , Hemo/metabolismo , Hemólisis , Hígado/enzimología , Proteínas de la Membrana/sangre , Alanina Transaminasa/sangre , Animales , Animales Recién Nacidos , Aspartato Aminotransferasas/sangre , Supervivencia Celular , Modelos Animales de Enfermedad , Eritroblastosis Fetal/sangre , Eritroblastosis Fetal/genética , Eritrocitos/efectos de los fármacos , Predisposición Genética a la Enfermedad , Hemo-Oxigenasa 1/deficiencia , Hemo-Oxigenasa 1/genética , Hemólisis/efectos de los fármacos , Hemopexina , Heterocigoto , Peroxidación de Lípido , Hígado/efectos de los fármacos , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Metemalbúmina/farmacología , Ratones , Ratones Noqueados , Células 3T3 NIH , Estrés Oxidativo , Fenotipo , Regiones Promotoras Genéticas , Unión Proteica , Factores de TiempoRESUMEN
Antioxidant, anti-inflammatory and anti-atherogenic effects have been associated with elevations of unconjugated bilirubin (UCB) in serum and with the induction of heme oxygenase-1 (HO-1), the rate-limiting enzyme in UCB synthesis. The aim of this study was to investigate the intracellular metabolism and antioxidant properties of UCB in human hepatoblastoma HepG2 cells and tissues of Wistar rats exposed to oxidative stressors and lipopolysaccharide (LPS), respectively. Intracellular UCB concentrations in HepG2 cells correlated with its levels in culture media (p < 0.001) and diminished lipid peroxidation in a dose-dependent manner (p < 0.001). Moreover, induction of HO-1 with sodium arsenite led to 2.4-fold (p = 0.01) accumulation of intracellular UCB over basal level while sodium azide-derived oxidative stress resulted in a 60% drop (p < 0.001). This decrease was ameliorated by UCB elevation in media or by simultaneous induction of HO-1. In addition, hyperbilirubinemia and liver HO-1 induction in LPS-treated rats resulted in a 2-fold accumulation of tissue UCB (p = 0.01) associated with enhanced protection against lipid peroxidation (p = 0.02). In conclusion, hyperbilirubinemia and HO-1 induction associated with inflammation and oxidative stress increase intracellular concentrations of UCB, thus enhancing the protection of cellular lipids against peroxidation. Therefore, the previously reported protective effects of hyperbilirubinemia and HO-1 induction are at least in part due to intracellular accumulation of UCB.
Asunto(s)
Antioxidantes/metabolismo , Bilirrubina/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Arsenitos/farmacología , Bilirrubina/administración & dosificación , Bilirrubina/análogos & derivados , Activación Enzimática/efectos de los fármacos , Hemo/análisis , Hemo-Oxigenasa 1/metabolismo , Células Hep G2 , Humanos , Hiperbilirrubinemia/metabolismo , Lipopolisacáridos/administración & dosificación , Metemalbúmina/farmacología , Ratas , Ratas Wistar , Compuestos de Sodio/farmacologíaRESUMEN
Although hypoxia induces heme oxygenase (HO)-1 protein and mRNA expression in many cell types, hypoxia has also been shown to decrease HO-1 mRNA and protein expression. We tested the hypothesis that 24-h preexposure to hypoxia in human placental preparations suppresses HO protein expression and enzymatic function. Immortalized HTR-8/SVneo first-trimester trophoblast cells and explants of normal human chorionic villi (CV) from term placentas were cultured for 24 h in 1%, 5%, or 20% O(2). HO protein levels were determined by Western blot analysis, and microsomal HO activity was measured. HO-2 protein content was decreased by 17% and 5% in human trophoblast cells after 24-h exposure to 1% and 5% O(2), respectively, versus 20% O(2). In contrast, HO-2 protein content in CV explants was unaffected by changes in oxygenation. HO-1 protein content, which was barely detectable in both biological systems, was not affected by changes in oxygenation. Similarly, HO enzymatic activity was unchanged in both preparations after 24-h exposure to 1%, 5%, or 20% O(2). The above data do not support the hypothesis that hypoxia in the human placenta suppresses both HO protein content and HO protein function. The present observations reinforce the necessity to determine both HO protein expression and function.
Asunto(s)
Hipoxia de la Célula/fisiología , Vellosidades Coriónicas/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Trofoblastos/metabolismo , Western Blotting , Línea Celular , Vellosidades Coriónicas/enzimología , Activación Enzimática/efectos de los fármacos , Femenino , Hemo , Hemo-Oxigenasa 1 , Humanos , Técnicas In Vitro , Proteínas de la Membrana , Metemalbúmina/farmacología , Microsomas/química , Microsomas/enzimología , Embarazo , Trofoblastos/citología , Trofoblastos/efectos de los fármacos , Trofoblastos/enzimologíaRESUMEN
Human monocytes avidly ingest malarial pigment, hemozoin. Phagocytosed hemozoin persists in the monocyte for a long time and modifies important monocyte functions. Stability of phagocytosed hemozoin may depend on modifications of the hemozoin heme moiety or reduced ability to express heme-inducible heme oxygenase. We show here that the spectral characteristics of alkali-solubilized hemozoin were identical to those of authentic heme, although hemozoin was solubilized by alkali much more slowly than authentic heme. Alkali-solubilized hemozoin was a substrate of microsomal rat heme oxygenase and bilirubin reductase, with bilirubin as the main final product. Hemozoin feeding to human monocytes did not induce heme oxygenase, but authentic heme and alkali-solubilized hemozoin supplemented to hemozoin-fed monocytes induced heme oxygenase and were degraded normally. Lysosomes isolated from hemozoin-fed monocytes released only traces of heme while lysosomes from erythrocyte-fed monocytes liberated considerable quantities of heme. Lack of heme release from hemozoin did not depend on proteolysis-resistant, heme-binding proteins, since lysosomal proteases fully degraded hemozoin-associated proteins but did not solubilize hemozoin. In conclusion, our data indicate that lack of induction of HO1 is due to the intrinsic structural characteristics of hemozoin and not to hemozoin-mediated impairment of the mechanism of HO1 induction.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/biosíntesis , Hemoproteínas/metabolismo , Monocitos/enzimología , Plasmodium falciparum/fisiología , Animales , Inducción Enzimática , Eritrocitos/parasitología , Glutatión/metabolismo , Hemo/metabolismo , Hemoproteínas/química , Hemoproteínas/farmacología , Humanos , Lisosomas/metabolismo , Metemalbúmina/farmacología , FagocitosisRESUMEN
Heme oxygenase in the liver microsomes of tadpole and adult bullfrog, Rana catesbeiana, was studied in relation to hemoglobin metabolism during tadpole development. The results obtained are as follows. 1. The specific activity of the enzyme of premetamorphic tadpole liver was comparable to that of adult frog liver. The apparent Km for methemalbumin was about 25 microM for both tadpole and frog enzymes. 2. The enzyme activity was stimulated in vivo by the injection of methemalbumin, phenylhydrazine and triiodothyronine to the animals. 3. A marked increase in the enzyme activity was found in the liver of tadpole during prometamorphic stage and metamorphic climax.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/metabolismo , Microsomas Hepáticos/enzimología , Oxigenasas de Función Mixta/metabolismo , Rana catesbeiana/metabolismo , Animales , Dactinomicina/farmacología , Larva/enzimología , Larva/crecimiento & desarrollo , Metemalbúmina/farmacología , Fenilhidrazinas/farmacología , Rana catesbeiana/crecimiento & desarrollo , Triyodotironina/farmacologíaRESUMEN
The aim of this work was to study possible uptake of electron-dense marker particles and uptake and degradation of proteins in vitro by two types of lysosomes, namely, secondary lysosomes and iron-laden "residual" bodies. The sedimentability and latency of lysosomal enzymes were measured to evaluate whether or not the different lysosomes were suitable for in vitro incubation experiments. Residual bodies displayed leakage and enzyme inactivation with increasing incubation times, whereas secondary lysosomes were stable. The two types of lysosomes were incubated in vitro with isotopically labeled methemoglobin or insulin. Secondary lysosomes, but not iron-laden residual bodies, were active in the degradation of the exogenously added proteins. Control experiments were performed to evaluate possible extra-lysosomal proteolysis of the substrates due to leakage of lysosomal enzymes during incubation. The enzymes released during incubation could only account for a minor portion of the degradation of the added labeled proteins. Secondary lysosomes were preincubated with radiolabeled protein, followed by washing and reincubation. The radioactivity in the lysosomal pellet decreased during reincubation concomitant with an increase in trichloroacetic acid-soluble label. Chloroquine decreased the release of degradation products from the lysosomes. Further support for the interpretation that lysosomes take up proteins was gained from ultrastructural observations. Secondary lysosomes were incubated with electron-dense Percoll particles in vitro. Morphologic analysis revealed Percoll particles inside intralysosomal vesicles which appeared to be formed during incubation. The results indicate that lysosomes are able to internalize their membranes during in vitro incubation followed by degradation of the micropinocytosed proteins.
Asunto(s)
Autofagia , Membranas Intracelulares/metabolismo , Hígado/metabolismo , Lisosomas/metabolismo , Fagocitosis , Proteínas/metabolismo , Fosfatasa Ácida/análisis , Animales , Catepsina D , Catepsinas/análisis , Fraccionamiento Celular , Cloroquina/farmacología , Técnicas In Vitro , Insulina/farmacología , Lisosomas/ultraestructura , Metemalbúmina/farmacología , Pinocitosis , RatasAsunto(s)
Compuestos Férricos/farmacología , Compuestos Ferrosos/farmacología , Hemoproteínas/farmacología , Hierro/farmacología , Microsomas Hepáticos/metabolismo , Estirenos/metabolismo , Animales , Ácido Edético/farmacología , Hemina/farmacología , Peróxido de Hidrógeno/farmacología , Masculino , Metemalbúmina/farmacología , Metahemoglobina/farmacología , Microsomas Hepáticos/efectos de los fármacos , Oxidación-Reducción , RatasRESUMEN
Delayed cerebral vasospams is caused by excessive accumulation of dopamine-beta-hydroxylase (DBH) and noradrenaline in cerebral vessel walls. This study demonstrates the mechanisms of delayed spasm, particularly the role of red blood cell components, and the successful relief of delayed cerebral vasospasm. Spasmogenic substances which contained a heme component, such as methemoglobin, methemalbumin and catalase enhanced DBH activity in human serum as measured by a one step chemical spectrophotometric assay. The concentration which gave the highest DBH activity caused the maximum constriction of the basilar artery, when the substances were applied topically. Among components of red cells, methemoglobin, methemalbumin, catalase and nicotinamid adenin dinucleotide (NADH) caused constriction of basilar artery in cats, when applied topically, whereas hematin, hemin and bilirubin caused no significant spasm. An oxyhemoglobin solution obtained by mixture with methemoglobin and ascorbic acid produced no significant vascular spasm either. Relief of delayed cerebral vasospasm was obtained with topical application of specific alpha adrenergic blocking drug such as phenoxybenzamine, specific inhibitors of DBH such as fusaric acid, o-phenanthroline and alphaalpha' dipyridyl beta2 adrenergic stimulants such as salbutamol, and a phosphodiesterase inhibitor, ascorbic acid.