RESUMEN
Isolated hepatocytes and liver microsomes incubated with monomethyl-1,1 dimethyl- and 1,2 dimethyl-hydrazines produced free radical intermediates which were detected by ESR spectroscopy by using 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) as spin trapping agent. The spectral features of the spin adducts derived from all three hydrazine derivatives corresponded to the values reported for the methyl free radical adduct of 4-POBN. In the microsomal preparations inhibitors of the mixed function oxidase system and the destruction of cytochrome P450 by pretreating the rats with CoCl2 all decreased the free radical formation. Methimazole, an inhibitor of FAD-containing monoxygenase system, similarly decreased the activation of 1,1 dimethyl-hydrazine, but not that of monomethyl- and 1,2 dimethyl-hydrazines. The addition to liver microsomes of physiological concentrations of glutathione (GSH) lowered by approx. 80% the intensities of the ESR signals. Consistently, incubation of isolated hepatocytes with methyl-hydrazines decreased the intracellular GSH content, suggesting that GSH can effectively scavenge the methyl free radicals. The results obtained suggest that methyl free radicals could be the alkylating species responsible for the toxic and/or carcinogenic effect of methyl-hydrazines.
Asunto(s)
Dimetilhidrazinas/farmacocinética , Hígado/metabolismo , Metilhidrazinas/farmacocinética , Microsomas Hepáticos/metabolismo , Monometilhidrazina/farmacocinética , Animales , Biotransformación , Inhibidores Enzimáticos del Citocromo P-450 , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Glutatión/farmacología , Hígado/efectos de los fármacos , Masculino , Microsomas Hepáticos/efectos de los fármacos , Oxigenasas de Función Mixta/antagonistas & inhibidores , Óxidos de Nitrógeno , Piridinas , Ratas , Ratas Endogámicas , Marcadores de SpinRESUMEN
The incubation of isolated rat hepatocytes with 0.172 mM carbon tetrachloride caused a rapid decrease in the calcium content of both mitochondrial and extramitochondrial compartments. However, the release of Ca2+ from the intracellular stores was not associated with an increase in the cytosolic Ca2+ levels as measured by activation of phosphorylase alpha or by Quin-2 fluorescence. A rapid rise in hepatocyte free calcium was only observed with concentrations of CCl4 higher than 0.172 mM. The lack of activation of phosphorylase alpha was not due to the inhibition of the enzyme by CCl4, since in CCl4-treated hepatocytes the phosphorylase activity could be stimulated by glucagon, butyryl--cAMP or by the increase of cell calcium induced by the addition of A23187. Ca2+-dependent ATPase of plasma membranes was only slightly affected in the early phases of poisoning with CCl4 when both mitochondrial and extramitochondrial calcium pools were already lowered. This led to the conclusion that calcium released from intracellular organelles could be extruded from the cells in sufficient amounts to prevent the increase of the cytosolic levels. A rise in hepatocyte free calcium was observed during the second hour of incubation with CCl4, concomitantly with the appearance of both LDH leakage and plasma membrane blebbing. The addition of EGTA to the medium prevented both the increase in cytosolic Ca2+ and the blebbing suggesting that they were a consequence of an influx of calcium into the cells. However, neither EGTA nor the addition of inhibitors of calcium-dependent phospholipase A2 or non-lysosomal proteases were able to protect against cell death. These latter results suggested that the alterations of calcium distribution induced by CCl4 in isolated hepatocytes were not a primary cause of the toxic effects, although they did not exclude that a sustained rise in cytosolic Ca2+ could contribute in the progression of cell injury.
Asunto(s)
Calcio/metabolismo , Tetracloruro de Carbono/farmacología , Homeostasis/efectos de los fármacos , Hígado/efectos de los fármacos , Animales , Activación Enzimática/efectos de los fármacos , Técnicas In Vitro , Hígado/citología , Hígado/enzimología , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Fosforilasa a/metabolismo , Ratas , Ratas EndogámicasRESUMEN
Incubation of rat liver microsomes with 1-propanol and 1-butanol in the presence of NADPH and of the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) allowed the detection of free radical intermediates tentatively identified as 1-hydroxypropyl and 1-hydroxybutyl radical, respectively. Microsomes isolated from rats treated chronically with ethanol (EtOH) or with the combination of starvation and acetone treatment (SA), exhibited a two-fold increase in the ESR signal intensity as compared to untreated controls, whereas no increase was observed in phenobarbital-induced (PB) microsomes. Consistently, in reconstituted membrane vesicles, ethanol-inducible cytochrome P450IIE1 was twice as active as phenobarbital-inducible P450IIB1 in producing 1-butanol free radicals. In the microsomal preparations from EtOH and SA pretreated rats the addition of antibodies against cytochrome P450IIE1, but not of preimmune IgGs, lowered the ESR signal of 1-butanol radicals by more than 50%. The same antibodies decreased the free radical production by untreated microsomes by 35-40%, but were ineffective on microsomes from PB-treated animals. This indicated that cytochrome P450IIE1 is the major enzyme responsible for the free radical activation of alcohols in control and ethanol-fed rats. The generation of 1-hydroxybutyl radicals by EtOH microsomes was inhibited by 40, 48 and 68%, respectively, by the addition of isoniazid, tryptamine and octylamine, compounds known to specifically affect the NADPH oxidase activity of this isoenzyme. This effect was not due to the scavenging of the alcohol radical since none of these compounds affected the ESR signals originated from 1-butanol in a xanthine-xanthine oxidase system. When added to reconstituted membrane vesicles isoniazid, tryptamine and octylamine also decreased 1-butanol radical formation by P450IIE1 by 54, 38 and 66%, respectively. Such an inhibition corresponded to the effect exerted by the same compounds on O2- release from P450IIE1 containing vesicles. These results indicate that the capacity of cytochrome P450IIE1 to reduce oxygen is related to its ability to generate alcohol free radicals and suggest that ferric cytochrome P450-oxygen complex might act as oxidizing species toward alcohols.
Asunto(s)
Alcoholes/metabolismo , Sistema Enzimático del Citocromo P-450/biosíntesis , Etanol/farmacología , Oxidorreductasas N-Desmetilantes/biosíntesis , 1-Butanol , Animales , Butanoles/metabolismo , Citocromo P-450 CYP2E1 , Sistema Enzimático del Citocromo P-450/metabolismo , Inducción Enzimática , Radicales Libres , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas , Oxidorreductasas N-Desmetilantes/metabolismo , Ratas , Ratas EndogámicasRESUMEN
Liver microsomes incubated with a NADPH regenerating system, ethanol and the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) produced an electron spin resonance (ESR) signal which has been assigned to the hydroxyethyl free radical adduct of 4-POBN by using 13C-labelled ethanol. The free radical formation was dependent upon the activity of the microsomal monoxygenase system and increased following chronic feeding of the rats with ethanol. The production of hydroxyethyl free radicals was stimulated by the addition of azide, while catalase and OH. scavengers decreased it. This suggested that hydroxyl radicals (OH.) produced in a Fenton-type reaction from endogenously formed hydrogen peroxide were involved in the free radical activation of ethanol. Consistently, the supplementation of iron, under various forms, also increased the intensity of the ESR signal which, on the contrary, was inhibited by the iron-chelating agent desferrioxamine. Microsomes washed with a solution containing desferrioxamine and incubated in a medium treated with Chelex X-100 in order to remove contaminating iron still produced hydroxyethyl radicals, although at a reduced rate. Under these conditions the free radical formation was apparently independent from the generation of OH. radicals, whereas addition of cytochrome P-450 inhibitors decreased the hydroxyethyl radical formation, suggesting that a cytochrome P-450-mediated process might also be involved in the activation of ethanol. Reduced glutathione (GSH) was found to effectively scavenge the hydroxyethyl radical, preventing its trapping by 4-POBN. The data presented suggest that ethanol-derived radicals could be generated during the microsomal metabolism of alcohol probably through two different pathways. The detection of ethanol free radicals might be relevant in understanding the pathogenesis of the liver lesions which are a consequence of alcohol abuse.
Asunto(s)
Etanol/metabolismo , Microsomas Hepáticos/metabolismo , Óxidos de Nitrógeno , Animales , Quelantes/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Deferoxamina/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Compuestos Ferrosos/metabolismo , Radicales Libres , Glutatión/metabolismo , Peróxido de Hidrógeno/metabolismo , Hidróxidos/metabolismo , Radical Hidroxilo , Hierro/metabolismo , Masculino , Microsomas Hepáticos/efectos de los fármacos , NADP/metabolismo , Piridinas , Ratas , Ratas Endogámicas , Resinas Sintéticas , Marcadores de SpinRESUMEN
Hydrazine derivatives constitute a wide group of compounds and have found application in industry, agriculture, and (as therapeutical agents) medicine. In spite of their widely spread use, several hydrazine derivatives are known to exert hepatotoxic effects and are carcinogenic. Free radical species are produced during the hepatic biotransformation of alkylhydrazines by both rat and humans liver microsomes. Cytochrome P-450 dependent monoxygenase system is responsible for the production of these reactive species and specific cytochrome P-450 isoenzymes appear to catalyze the formation of free radical intermediates. Free radicals generated during the metabolism of alkylhydrazines are capable of inducing oxidative stress in isolated hepatocytes and might contribute to the development of cell injury.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Hidrazinas/toxicidad , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , Animales , Radicales Libres/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/enzimología , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Oxidación-Reducción , RatasRESUMEN
By using e.s.r. spectroscopy coupled with the spin trapping technique we have detected the formation of free radical intermediates by rat liver microsomes incubated with either ethanol, 2-propanol or 2-butanol in the presence of a NADPH regenerating system and 4-pyridyl-l-oxide-t-butyl nitrone (4-POBN) as spin trap. The e.s.r. spectra have been identified as due to the hydroxyalkyl free radical adducts of 4-POBN. The free radical formation depends upon the activity of the microsomal monoxygenase system and is blocked by omitting NADP+ from the incubation mixture, by anaerobic incubation or by enzyme denaturation. The involvement of hydroxyl radicals (OH.) produced through a Fenton-type reaction from endogenously formed hydrogen peroxide is suggested by the opposite effects exerted on the e.s.r. signal intensity by azide and catalase. Consistently, iron chelation by desferrioxamine inhibits the free radical formation, while the supplementation of EDTA-iron increases it by several fold. Inhibitors of cytochrome P450-dependent monoxygenase system reduce to various extents the production of free radical intermediates suggesting that reactive oxygen species might be formed at the active site of cytochrome P450 where they react with alkyl alcohol molecules. The data presented support the hypothesis that free radical species are generated during the microsomal metabolism of alcohols and suggest the possibility that ethanol-derived radicals might play a role in the pathogenesis of the liver lesions consequent upon alcoholic abuse.
Asunto(s)
1-Propanol/metabolismo , Butanoles/metabolismo , Etanol/metabolismo , Microsomas Hepáticos/metabolismo , Animales , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Masculino , Ratas , Ratas EndogámicasRESUMEN
Electron spin resonance (ESR) analysis combined with the use of 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) and dibromonitroso benzenesulfonic acid (DBNBS) as spin-trapping agents was used to characterize free radical generation during the metabolism of the anticancer agent procarbazine [N-isopropyl-a-(2-methylhydrazino)-p-toluamide hydrochloride]. The formation of free radical species, identified as methyl radicals, was observed during oxidation of procarbazine in rat liver microsomes and isolated hepatocytes in vitro, as well as in several organs following administration of the drug in vivo. A cytochrome P450-mediated reaction, involving P450IA and IIB isoenzymes, was responsible for the activation process. The metabolic pathway leading to free radical formation was characterized using various procarbazine metabolites and revealed strict analogies with previously published data on methane production from procarbazine. These results supported the identification of the trapped species as methyl free radical and suggested that C-oxidation of azoprocarbazine is the main source of radical intermediates derived from this anticancer drug.