RESUMEN
Thiol oxidation by hypochlorous acid and chloramines is a favorable reaction and may be responsible for alterations in regulatory or signaling pathways in cells exposed to neutrophil oxidants. In order to establish the mechanism for such changes, it is necessary to appreciate whether these oxidants are selective for different thiols as compared with other scavengers. We have measured rate constants for reactions of amino acid chloramines with a range of thiols, methionine, and ascorbate, using a combination of stopped-flow and competitive kinetics. For HOCl, rate constants are too fast to measure directly by our system and values relative to reduced glutathione were determined by competition with methionine. For taurine chloramine, the rate constants for reaction with 5-thio-2-nitrobenzoic acid, GSH, methionine, and ascorbate at pH 7.4 were 970, 115, 39, and 13 M(-1) s(-1), respectively. Values for 10 thiols varied by a factor of 20 and showed an inverse relationship to the pK(a) of the thiol group. Rate constants for chloramines of glycine and N-alpha-acetyl-lysine also showed these relationships. Rates increased with decreasing pH, suggesting a mechanism involving acid catalysis. For hypochlorous acid, rates of reaction with 5-thio-2-nitrobenzoic acid, GSH, cysteine, and most of the other thiols were very similar. Relative reactivities varied by less than 5 and there was no dependence on thiol pK(a). Chloramines have the potential to be selective for different cellular thiols depending on their pK(a). For HOCl to be selective, other factors must be important, or its reactions could be secondary to chloramine formation.
Asunto(s)
Aminoácidos/metabolismo , Cloraminas/metabolismo , Ácido Hipocloroso/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Ácido Ascórbico/metabolismo , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Metionina/metabolismo , Oxidantes/metabolismo , Oxidación-ReducciónRESUMEN
A program for thorough complex examination of individuals who liquidated Chernobyl power accident consequences provides objective diagnostic information and early diagnosis of both general and oncologic diseases. The program covers examination in regional Medical Centers giving highly qualified medical care for the liquidators. Regional and county hospitals can participate in the program, concerning also individuals exposed to radiation during other radiation accidents.
Asunto(s)
Enfermedades Profesionales/diagnóstico , Servicios de Salud del Trabajador/organización & administración , Liberación de Radiactividad Peligrosa , Adulto , Humanos , Persona de Mediana Edad , Enfermedades Profesionales/etiología , Enfermedades Profesionales/prevención & control , Salud Laboral/legislación & jurisprudencia , UcraniaRESUMEN
A simple and reproducible microtiter plate assay for measuring superoxide dismutase (SOD) activity is described. Water-soluble tetrazolium, the sodium salt of 4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-be nzene disulfonate, was used as a detector of superoxide radical generated by xanthine oxidase and hypoxanthine, in the presence of a range of concentrations of superoxide dismutase. A major advantage of the assay is that one reaction mixture is prepared and aliquotted into wells, avoiding pipetting errors and variable xanthine oxidase activity between samples. Inclusion of standardized SOD solution in each run enables inter-assay comparability without requiring a constant superoxide generation rate under all occasions. The assay is applicable for chloroform-ethanol red cell extracts as well as tissue homogenates without high-speed centrifugation. Fifty percent inhibition of formazan formation was achieved at 2.4+/-0.1 ng of Cu, ZnSOD per well with the coefficient of variation 4.2%.
Asunto(s)
Superóxido Dismutasa/análisis , Sales de Tetrazolio/química , Animales , Eritrocitos/enzimología , Humanos , Hipoxantina/análisis , Indicadores y Reactivos , Hígado/enzimología , Miocardio/enzimología , Ratas , Ratas Wistar , Superóxidos/análisis , Xantina Oxidasa/análisisRESUMEN
Using the catechol Tiron as an O2-. scavenger, we showed that sea sponges (Sycon sp.) produce superoxide radicals in sea water at a high rate without any stimuli added. The rate of O2-. outflow from sponges to their water surroundings reaches a value of 0.5 nmol/min per sponge at pH 6.5. The generation of O2-. was inhibited by Cu,Zn-superoxide dismutase, and restored by the addition of KCN. We also confirmed the abiotic production of O2-. in sea water, detected earlier with a different method by Petasne and Zika [Nature 325 (1987) 516-518].
Asunto(s)
Poríferos/metabolismo , Superóxidos/metabolismo , Animales , Agua de MarRESUMEN
Isoenzyme profiles and thermal stability of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) of clawed frogs of genus Xenopus (X. laevis and X. borealis) were compared. Earlier the presence of an unusually thermolabile mutant Cu,Zn-SOD as well as normal enzyme was shown in X. laevis. Having confirmed the data, we further show that X. borealis contains only the thermolabile Cu,Zn-SOD. This suggested that a mutation in the Cu,Zn-SOD gene and genome duplication have occurred in the immediate predecessor of X. laevis. Later on the parent Cu,Zn-SOD gene was lost, leaving only the mutated form of Cu,Zn-SOD in X. borealis.
Asunto(s)
Isoenzimas/metabolismo , Superóxido Dismutasa/metabolismo , Xenopus laevis/metabolismo , Xenopus/metabolismo , Animales , Estabilidad de Enzimas , Evolución Molecular , Humanos , Isoenzimas/genética , Mutación , Filogenia , Especificidad de la Especie , Superóxido Dismutasa/genética , Temperatura , Xenopus/genética , Xenopus laevis/genéticaRESUMEN
It is well established that superoxide dismutase (SOD) is the irresplaceable enzyme for aerobic lifestyle. Our understanding of its role has made strides recently as the result of gene transfection approach. Available data on consequences of Cu,Zn-SOD gene transfection in cell resistance to oxygen toxicity are reviewed. There are data that increasing only Cu,Zn-SOD can be toxic, and the balance between Cu,Zn-SOD and peroxide-removing enzymes is supposed to be of prime importance in the antioxidant defence. Role of Cu,Zn-SOD deregulation in carcinogenesis is discussed.
Asunto(s)
Dosificación de Gen , Estrés Oxidativo , Superóxido Dismutasa/genética , Animales , HumanosRESUMEN
Data on direct and indirect effects of reactive oxygen species on DNA are reviewed. The classification of the types of DNA oxidative damage and repair enzymes including glycosylases and endo- and exonucleases is presented. The regulatory function of reactive oxygen species and their role in carcinogenesis are discussed.
Asunto(s)
ADN/química , Especies Reactivas de Oxígeno , Daño del ADN , Reparación del ADN , Neoplasias/genética , Neoplasias/patología , Estrés OxidativoRESUMEN
Nuclear DNA damage resulting from incubation of isolated rat liver nuclei in the presence of NADPH or NADH and ethylenediamine tetraacetic acid, or diethylenetriamine pentaacetic acid, or desferoxamine complexes with Fe3+, has been studied. The degree of DNA damage was assessed by agarose electrophoresis. The observed damage was prevented by catalase but not superoxide dismutase. The mechanism of oxidative damage of nuclear DNA during membrane redox chains functioning is discussed.
Asunto(s)
Núcleo Celular/metabolismo , Daño del ADN , Especies Reactivas de Oxígeno , Animales , Catalasa/metabolismo , Ácido Edético , Radicales Libres , Hígado/metabolismo , NAD/metabolismo , NADP/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Ratas , Superóxido Dismutasa/metabolismoRESUMEN
Nuclear DNA damage, as the result of active oxygen formation by NAD(P)H-dependent redox chains, was studied. Isolated rat liver nuclei were incubated in the presence of NAD(P)H and iron chelators. Nuclear DNA damage was analyzed by electrophoresis in alkaline agarose. DNA damage after the addition of electron donors alone or with FeCl3 or DFO-Fe3+ was not visualized. Dramatic decay of high molecular weight DNA was observed with EDTA-Fe3+ or DTPA-Fe3+ in the presence of NAD(P)H. SOD did not prevent DNA damage, whereas catalase was protective. DNA damage was revealed after the addition of cumene hydroperoxide with EDTA-Fe3+, and it was sharply increased in the presence of NADPH. It is suggested that alkoxyl radicals in addition to hydroxyl radicals are involved in DNA damage during NAD(P)H oxidation in the presence of iron chelators, which can be reduced by membrane redox chains.
Asunto(s)
Núcleo Celular/metabolismo , Daño del ADN , NADP/metabolismo , NAD/metabolismo , Animales , Derivados del Benceno/farmacología , Quelantes/farmacología , ADN/aislamiento & purificación , Ácido Edético/farmacología , Hígado/metabolismo , Modelos Biológicos , Oxidación-Reducción , Ácido Pentético/farmacología , RatasRESUMEN
Environmental changes affect the active oxygen generation and thereby involve the oxidative stress. Estimation of the active oxygen metabolism as a method of biotesting is discussed. The results of measurement of specific superoxide dismutase suggest that this approach may be used for environmental assessment.
Asunto(s)
Monitoreo del Ambiente , Estrés Oxidativo , Animales , Biomarcadores/análisis , Carcinógenos Ambientales/toxicidad , ADN/efectos de los fármacos , Daño del ADN , Humanos , Oxígeno/toxicidad , Superóxido Dismutasa/análisisRESUMEN
An intense proteolytic degradation of both proteins and phosphoproteins has been observed in isolated nuclear matrices from rat liver, Zajdela Hepatoma and Hepatoma 22a, incubated with NP-40, DTT and gamma-[32P] ATP being most intense in Hepatoma 22a. Practically all phosphoproteins of Hepatoma 22a nuclear matrix degraded. This implies either an extremely high proteolytic activity in the preparation or the presence of a specific to phosphoproteins protease absent from rat liver and Zajdela Hepatoma nuclear matrices.
Asunto(s)
Neoplasias Hepáticas Experimentales/enzimología , Hígado/enzimología , Matriz Nuclear/enzimología , Proteínas Nucleares/metabolismo , Péptido Hidrolasas/metabolismo , Fosfoproteínas/metabolismo , Adenosina Trifosfato/farmacología , Animales , Autorradiografía , Electroforesis , Técnicas In Vitro , Ratones , Ratones Endogámicos C3H , RatasRESUMEN
The sensitivity of three human fibroblast lines, trisomic with respect to chromosome 21, to an anthracycline antibiotic carminomycin was compared with that of a normal fibroblast line using a 51Cr release assay. It was found that for an intermediate antibiotic concentration (10 microM) the sensitivity of trisomic fibroblasts, of increased content of Cu,Zn-superoxide dismutase was lower. These results suggest a role for superoxide-mediated membrane damage in the cytotoxic action of anthracycline antibiotics.
Asunto(s)
Carubicina/farmacología , Cromo/metabolismo , Daunorrubicina/análogos & derivados , Síndrome de Down/metabolismo , Superóxido Dismutasa/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Síndrome de Down/enzimología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/metabolismo , HumanosRESUMEN
O2-. generation by the succinate oxidase segment of the respiratory chain of mitochondria and submitochondrial particles from hepatoma 22a and hepatoma Zajdela has been studied with the use of the Tiron method. In the presence of succinate, superoxide generation is induced by antimycin, 2-n-4-hydroxyquinoline N-oxide or funiculosin, and is inhibited by mucidin, myxothiazol or cyanide. The rate of O2-. generation in the antimycin-inhibited state is maximal at the [succinate]/[fumarate] ratio of 1:10 and diminishes at more positive and more negative redox potentials. These characteristics of O2-.-generation are the same as observed earlier in submitochondrial particles from normal tissues. Accordingly, the mechanism of superoxide production is suggested to be the same in tumor and normal mitochondria, namely, autoxidation of the unstable ubisemiquinone in the ubiquinol-oxidizing centre o of cytochrome bc1 complex. With respect to the rate of O2-. generation, the hepatoma mitochondrial membranes are approximately twice as active as bovine heart submitochondrial particles and exceed those from rat liver by more than one order of magnitude.
Asunto(s)
Benzoquinonas , Neoplasias Hepáticas Experimentales/metabolismo , Mitocondrias/metabolismo , Superóxidos/metabolismo , Sal Disódica del Ácido 1,2-Dihidroxibenceno-3,5-Disulfónico , Animales , Antraquinonas/farmacología , Antimicina A/análogos & derivados , Antimicina A/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón/efectos de los fármacos , Radicales Libres , Hidroxiquinolinas/farmacología , Cinética , Masculino , Ratones , Ratones Endogámicos C3H , Mitocondrias/efectos de los fármacos , Oxidación-Reducción , Oxidorreductasas/metabolismo , Quinonas/metabolismo , Ratas , Partículas Submitocóndricas/metabolismo , Succinatos/farmacología , Ácido SuccínicoRESUMEN
Human erythrocyte membranes catalyse the NAD(P)H-dependent generation of the semiquinone of an adriamycin-type antibiotic carminomycin under anaerobic conditions. The maximal yield of the antibiotic radical is about 4-fold higher in the presence of NADPH than of NADH. The possible significance of the antibiotic reduction to the semiquinone by a human erythrocyte membrane redox chain for the clinical usage of these antibiotics is discussed.
Asunto(s)
Carubicina/sangre , Daunorrubicina/análogos & derivados , Membrana Eritrocítica/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Electrones , Radicales Libres , Humanos , Técnicas In Vitro , Cinética , NAD/farmacología , NADP/farmacología , Oxidación-ReducciónRESUMEN
Nuclear membranes from many tumors contain an unusual redox chain discovered originally in the Hepatoma 22a nuclear membranes which catalyzes superoxide dismutase-sensitive adrenaline oxidation to adrenochrome in the presence of either NADPH or NADH as electron donor, the reaction being inhibited by cyanide and azide. This redox chain can reduce anthracycline antitumor antibiotics adriamycin and carminomycin to their free radical states under anaerobic conditions. Evidence has been obtained for a higher stability of the carminomycin radical as compared to that of adriamycin. Operation of the nuclear membrane-bound redox chain can be a source of oxygen radical-mediated single strand breaks in DNA. The role of the nuclear membrane-associated electron transfer chain in augmenting the anticancer action of the anthracycline antibiotics is discussed.
Asunto(s)
Neoplasias Hepáticas Experimentales/metabolismo , NADP/metabolismo , Superóxidos/metabolismo , Animales , Carubicina/metabolismo , Carubicina/farmacología , Núcleo Celular/metabolismo , Daño del ADN , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Radicales Libres , Membranas Intracelulares/metabolismo , Oxidación-Reducción , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Xanthine oxidase reduces carminomycin and adriamycin to the semiquinones which have been detected by ESR technique. The steady state carminomycin semiquinone concentration is some tens times higher than the corresponding value for adriamycin. This effect appears to be a result of carminomycin semiquinone stabilization due to internal hydrogen bonding.
Asunto(s)
Carubicina/metabolismo , Daunorrubicina/análogos & derivados , Doxorrubicina/metabolismo , Xantina Oxidasa/metabolismo , Carubicina/efectos adversos , Doxorrubicina/efectos adversos , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Técnicas In VitroRESUMEN
Rat liver nuclei generate superoxide radicals in the presence of NADPH. Active oxygen species induced nicks in nuclear DNA. This was prevented by superoxide dismutase and catalase as well as by anaerobiosis. EDTA-Fe3+ dramatically increased the active oxygen-dependent DNA nicking.
Asunto(s)
Núcleo Celular/metabolismo , ADN-Topoisomerasas de Tipo I/biosíntesis , Superóxidos/metabolismo , Animales , ADN Polimerasa Dirigida por ADN/metabolismo , Ácido Edético/farmacología , Electroforesis en Gel de Agar , Radicales Libres , Peróxido de Hidrógeno/metabolismo , Hierro/farmacología , Hígado/metabolismo , NADP/metabolismo , Oxígeno/metabolismo , Ratas , Ratas EndogámicasRESUMEN
It has been discovered that nuclear membranes isolated from human brain tumors are capable of cyanide-sensitive oxidation of adrenaline to adrenochrome in the presence of both NAD X H and NADP X H while the process of oxidation is inhibited by superoxide dismutase. At the same time nuclear membranes isolated from the brain tissue do not oxidize adrenaline to adrenochrome in the presence of both substrates. A possible relationship between the properties of the nuclear membranes and the degree of tumor malignancy is discussed.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Membrana Nuclear/metabolismo , Superóxidos/metabolismo , Adrenocromo/metabolismo , Encéfalo/metabolismo , Epinefrina/antagonistas & inhibidores , Epinefrina/metabolismo , Humanos , Técnicas In Vitro , NADP/antagonistas & inhibidores , NADP/metabolismo , Oxidación-Reducción , Superóxido Dismutasa/metabolismoRESUMEN
Evidence has been obtained that the NAD(P)H-dependent "generation of superoxide radicals" by various types of membrane bound redox chains, as studied by the adrenaline method, does not occur in the absence of adrenaline. Studies of the oxygen uptake associated with the NAD(P)H-dependent adrenaline co-oxidation confirm the presence of an unusual cyanide-sensitive electron transfer system in the nuclear membranes from Hepatoma 22a. This redox chain contains a b-type cytochrome which resembles cytochrome b5.