RESUMEN
Previous work suggested that arsine- (AsH3-) induced hemoglobin (HbO2) damage may lead to hemolysis (Hatlelid et al., 1996). The purpose of the work presented here was to determine whether reactive oxygen species are formed by AsH3 in solution, in hemoglobin solutions, or in intact red blood cells, and, if so, to determine whether these species are responsible for the observed hemoglobin damage. Hydrogen peroxide (H2O2) was detected in aqueous solutions containing AsH3 and HbO2 or AsH3 alone but not in intact red blood cells or lysates. Additionally, high-activity catalase (19,200 U/ml) or glutathione peroxidase (68 U/ml) added to solutions of HbO2 and AsH3 had only a minor protective effect against AsH3-induced damage. Further, the differences between the visible spectra of AsH3-treated HbO2 and H2O2-treated HbO2 indicate that two different degradative processes occur. The presence of superoxide anion (O2-) was measured by O2(-)-dependent reduction of nitro blue tetrazolium (NBT). The results were negative for O2-. Exogenous superoxide dismutase (100 micrograms/ml) did not affect AsH3-induced HbO2 spectral changes, nor did the hydroxyl radical scavengers, mannitol, and DMSO (20 mM each). The general antioxidants ascorbate (< or = 10 mM) and glutathione (< or = 1 mM) also had no effect. These results indicate that the superoxide anion and the hydroxyl radical (OH) are not involved in the mechanism of AsH3-induced HbO2 damage. The results also indicate that although AsH3 contributes to H2O2 production in vitro, cellular defenses are adequate to detoxify the amount formed. An alternative mechanism by which an arsenic species is the hemolytic agent is proposed.
Asunto(s)
Contaminantes Ocupacionales del Aire/toxicidad , Intoxicación por Arsénico , Arsenicales , Hemoglobinas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Catalasa/farmacología , Relación Dosis-Respuesta a Droga , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Eritrocitos/patología , Depuradores de Radicales Libres/farmacología , Glutatión Peroxidasa/farmacología , Hemoglobinas/metabolismo , Hemólisis , Peróxido de Hidrógeno/análisis , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/toxicidad , Radical Hidroxilo/análisis , Radical Hidroxilo/metabolismo , Radical Hidroxilo/toxicidad , Masculino , Manitol/farmacología , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/farmacología , Superóxidos/análisis , Superóxidos/metabolismo , Superóxidos/toxicidad , Sales de Tetrazolio/metabolismoRESUMEN
The mechanism of arsine (AsH3) induced hemolysis was studied in vitro using isolated red blood cells (RBCs) from the rat or dog. AsH3-induced hemolysis of dog red blood cells was completely blocked by carbon monoxide (CO) preincubation and was reduced by pure oxygen (O2) compared to incubations in air. Since CO and O2 bind to heme and also reduce hemolysis, these results suggested a reaction between AsH3 and hemoglobin in the heme-ligand binding pocket or with the heme iron. Further, sodium nitrite induction of methemoglobin (metHb) to 85% and 34% of total Hb in otherwise intact RBCs resulted in 56% and 16% decreases in hemolysis, respectively, after incubation for 4 h. This provided additional evidence for the involvement of hemoglobin in the AsH3-induced hemolysis mechanism. Reactions between AsH3 and hemoglobin were studied in solutions of purified dog hemoglobin. Spectrophotometric studies of the reaction of AsH3 with various purified hemoglobin species revealed that AsH3 reacted with HbO2 to produce metHb and, eventually, degraded Hb characterized by gross precipitation of the protein. AsH3 did not alter the spectrum of deoxyHb and did not cause degradation of metHb in oxygen, but bound to and reduced metHb in the absence of oxygen. These data indicate that a reaction of AsH3 with oxygenated hemoglobin HbO2, may lead to hemolysis, but there are reactions between AsH3 and metHb that may not be directly involved in the hemolytic process.
Asunto(s)
Contaminantes Ocupacionales del Aire/metabolismo , Arsenicales/metabolismo , Hemoglobinas/metabolismo , Animales , Intoxicación por Arsénico , Arsenicales/química , Perros , Femenino , Hemoglobinas/química , Hemólisis/efectos de los fármacos , Masculino , Metahemoglobina/metabolismo , Ratas , Ratas Sprague-Dawley , EspectrofotometríaRESUMEN
A novel test system using isolated red blood cells (RBCs) and arsine (AsH3) in aqueous solution was developed to allow quantitation of AsH3 exposure and to study the toxicity of AsH3 in vitro. In this system AsH3 gas was generated and dissolved in aqueous solution, the concentration was measured, and the standardized solution was mixed with rat or dog red blood cells (RBCs). AsH3 was found to be stable in solution at neutral pH for several hours, but was lost quickly from solution as the acidity was increased to pH 2. Approximately 74% of the initial 0.56 mM AsH3, measured as total arsenic, was found to be taken up by, or strongly associated with, dog RBCs within 5 min of incubation and 82% of the initial 0.49 mM AsH3 was found in rat RBCs after 10 min incubation. Following hypotonic lysis of rat RBCs, 55% of the cell-associated arsenic was found in the membrane fraction with the balance found in the cytosolic fraction. The in vitro technique was used to examine factors influencing AsH3 toxicity using hemolysis as the end point. Hemolysis levels in dog and rat RBC incubations were found to increase with time after exhibiting a lag phase of about 30 min. At the AsH3 concentrations used, maximum levels of hemolysis were observed by 2 hr; maximum hemolysis at room temperature for dog RBCs was 20% and for rat RBCs was 22%. Increasing the temperature from room temperature to 37 degrees C resulted in increased hemolysis in dog RBCs (36%) and rat RBCs (90%).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Intoxicación por Arsénico , Eritrocitos/efectos de los fármacos , Hemólisis/efectos de los fármacos , Animales , Arsenicales/análisis , Perros , Femenino , Técnicas In Vitro , Masculino , Ratas , Ratas Sprague-Dawley , Compuestos de Sulfhidrilo/análisis , Factores de Tiempo , Pruebas de ToxicidadRESUMEN
Arsenic is metabolized by living systems using oxidation-reduction and methylation reactions, and reduced glutathione (GSH) has been shown to be important in that metabolism. In this study, the solution reactions between GSH and arsenate, arsenite, and their methylated metabolites, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), were characterized using 1H and 13C NMR under a nitrogen atmosphere. Binding to GSH through the thiol group was primarily followed by shifts in the carbon atom bonded to the sulfhydryl group of the cysteinyl residue, i.e., the CH2 carbon atom and the protons bonded to it. The methylated metabolites also showed shifts in the methyl groups attached to the arsenic atom after reaction with GSH. Sodium arsenite, As(III), bound to GSH to form an As(SG)3 complex in solution as indicated by NMR spectra. The identity of the complex was confirmed by FAB-MS after isolation of the compound. Mixtures of sodium arsenate, As(V), and GSH showed that arsenate oxidized GSH in D2O solutions at pH 7 to form oxidized glutathione (GSSG). When the molar ratio of As:GSH exceeded 1:2, evidence for the formation of As(SG)3 was observed. MMA and DMA are both As(V) species, and mixtures with GSH showed oxidation to GSSG initially followed by formation of CH3.As(SG)2 and (CH3)2.As.SG, respectively. The effects of GSH on arsenic metabolism may result from direct reactions between the two compounds.
Asunto(s)
Arsénico/química , Glutatión/química , Arseniatos/química , Arsenicales/síntesis química , Arsenicales/química , Ácido Cacodílico/química , Espectroscopía de Resonancia Magnética , Oxidación-Reducción , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
Irradiation of adenylate kinase (AK) from chicken muscle with 300-400-nm light in the presence of 0.25 mM vanadate ion first inactivated the enzyme and then cleaved the polypeptide chain near the NH2 terminus. The addition of the multisubstrate analogue, P1,P5-bis(5'-adenosyl) pentaphosphate, prevented both effects. ATP, but not AMP, blocked both inactivation and cleavage in a saturable manner, suggesting that both effects were due to modification at the ATP-binding site. The polypeptide products of the photocleavage were isolated by HPLC and characterized by amino acid composition, peptide sequencing, and mass spectral analyses. The predominant (greater than 90%) small peptide fragment contained the first 16 amino acids from the amino terminus of the enzyme. The amino terminus of this peptide contained an acetylated serine, and the "carboxy" terminus was modified by a cyclized gamma-aminobutyric acid which originated from photooxidation and decarboxylation of proline-17 by vanadate. Edman sequencing indicated that the majority of the large peptide fragment (Mr approximately 19,500) was amino-terminal blocked, but a small portion was sequenceable starting at either glycine-18 (7%) or serine-19 (2%). These studies indicate that in the ATP-AK complex proline-17 is close to the phosphate chain of ATP but not AMP, consistent with the latest evaluation of nucleotide-binding sites on mitochondrial matrix AK by X-ray crystallography [Diederichs, K., & Schulz, G.E. (1991) J. Mol. Biol. 217, 541-549]. Furthermore, this is the first report that an amino acid other than serine can be involved in vanadate-promoted photocleavage reactions.