RESUMO
Plasma and urinary levels of malondialdehyde-like products (MDA) and isoprostanes were identified as markers of in vivo lipid peroxidation in an animal model of CCl4 poisoning. We sought to determine the extent to which the formation of these oxidation products is influenced by inhibition of the cyclooxygenase enzymes which catalytically generate proinflammatory lipid peroxidation products known as prostaglandins and thromboxane. In the present studies, after induction of oxidant stress in rats with CCl4, lipid peroxidation products measured in plasma and urine demonstrate that isoprostanes and MDA can be partially inhibited by cyclooxygenase inhibitors, albeit to different extents. The lowering of isoprostane and MDA formation, however, may not to due primarily to the diminution of catalytic generation of isoprostanes or MDA by the cyclooxygenases but, rather, may be the result of the suppression of nonenzymatic lipid peroxidation. This is suggested since 8,12-iso-iPF2alpha-VI is also reduced by indomethacin, yet, unlike other isoprostanes and MDA, it is not generated catalytically by the cyclooxygenase. Thus, although the two cyclooxygenase inhibitors we tested have statistically significant effects on the measurements of both isoprostanes and MDA in this study, the results provide evidence that these lipid-degradation products primarily constitute markers of oxidative stress.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Biomarcadores/metabolismo , Intoxicação por Tetracloreto de Carbono/tratamento farmacológico , Tetracloreto de Carbono/toxicidade , Indometacina/farmacologia , Metabolismo dos Lipídeos , Ácido Meclofenâmico/farmacologia , Estresse Oxidativo , Animais , Cromatografia Líquida de Alta Pressão , Radicais Livres , Cromatografia Gasosa-Espectrometria de Massas , Imunoensaio , Indometacina/metabolismo , Inflamação , Peroxidação de Lipídeos , Espectrometria de Massas , Oxigênio/metabolismo , Prostaglandinas/metabolismo , Isoformas de Proteínas , Ratos , Ratos Endogâmicos F344 , Tromboxano A2/metabolismo , Fatores de TempoRESUMO
Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.
Assuntos
Intoxicação por Tetracloreto de Carbono/metabolismo , Tetracloreto de Carbono/toxicidade , DNA/metabolismo , Desoxiguanosina/análogos & derivados , Metabolismo dos Lipídeos , Estresse Oxidativo , 8-Hidroxi-2'-Desoxiguanosina , Animais , Ensaio Cometa , Dano ao DNA , Desoxiguanosina/farmacologia , Radicais Livres , Cromatografia Gasosa-Espectrometria de Massas , Peróxido de Hidrogênio/metabolismo , Imunoensaio , Immunoblotting , Fígado/metabolismo , Masculino , Malondialdeído/farmacologia , Metionina/metabolismo , Oxigênio/metabolismo , Ratos , Ratos Endogâmicos F344 , Espectrofotometria , Substâncias Reativas com Ácido Tiobarbitúrico , Fatores de Tempo , Tirosina/química , Tirosina/metabolismoAssuntos
Dano ao DNA , Reparo do DNA , DNA Mitocondrial , Animais , Radicais Livres , Humanos , Sobrecarga de Ferro , RatosRESUMO
In the autoxidation of 6-hydroxydopamine, we investigated the reactivity of metals and metal complexes with a range of abilities to catalyse the reaction with oxygen. Comparing the catalytic effectiveness of aquo metals at pH 7.4, copper accelerated autoxidation 61-fold, iron 24-fold, manganese 7.3-fold, and vanadium 5.7-fold. Copper was thus the most effective catalyst despite being the weakest oxidant, indicating reduction of oxygen as rate limiting. EDTA, which decreases the reduction potential of Fe(III)/Fe(II), increased catalysis by iron 74% to almost that of aquo copper. Conversely, EDTA inhibited catalysis by copper, manganese, and vanadium. Desferrioxamine strongly inhibited catalysis by all of the metals. Histidine prevented catalysis by copper, accelerated catalysis by iron (43%), and had little effect on catalysis by manganese or vanadium. ADP and phytate inhibited catalysis by iron and manganese (50% or more), accelerated catalysis by vanadium (10-27%), and had no effect on catalysis by copper. The effects of the ligands largely reflected their influence on the reduction potential of the metal. Accordingly, addition of NaBr, which increases the reduction potential of Cu(II)/Cu(I), inhibited by 50%. In contrast, Na2SO4 augmented catalysis by copper 3-fold. Consistent with effects of OH- on reduction potentials and on metal coordination to 6-hydroxydopamine, an increase in pH to 8.0 decreased catalysis by copper and iron, but increased that of manganese 10-fold. In conclusion, the catalytic effectiveness of the metal-ligand complexes are largely attributable to their reduction potential, with steric accessibility playing secondary roles. The results delineate a window of catalytically effective potentials suitable for facile reduction and reoxidation by oxygen. By extension the results identify factors determining the pro- and antioxidant roles of ligands in metal mediated reduction of oxygen.
Assuntos
Cobre/química , Ferro/química , Manganês/química , Oxidopamina/química , Oxigênio/química , Vanádio/química , Difosfato de Adenosina/química , Brometos/química , Soluções Tampão , Catálise , Desferroxamina/química , Ácido Edético/química , Histidina/química , Concentração de Íons de Hidrogênio , Ligantes , Concentração Osmolar , Oxirredução , Ácido Fítico/química , Compostos de Sódio/química , TermodinâmicaRESUMO
Numerous studies have shown that iron-loaded diets increase markers of lipid peroxidation in rats, but few have addressed the effects of oral iron supplements on these markers. We investigated the effects of daily and intermittent iron supplements on iron and vitamin E status, and lipid peroxidation. Iron supplements were administered in doses equivalent to those often given to pregnant women in the developing world. In Study 1, iron-deficient (D) and iron-normal (N) rats were fed either 0 or 8000 microgram of supplemental iron daily for 21 d. In Study 2, D rats were fed either the same supplements daily or once every 3 d (8 supplements total). Lipid peroxidation was assessed by breath ethane and pentane and by malondialdehyde (MDA) (using GC-MS). In Study 1, daily supplemented N and D rats had liver nonheme iron concentrations that were 1.8- and 2.7-fold higher, respectively, than those in unsupplemented N rats. Breath ethane levels were also higher in supplemented rats (P < 0.05), but MDA (in plasma, liver, kidney) and liver vitamin E did not differ. Unexpectedly, severely D, anemic rats had significant elevations in the levels of breath ethane, liver MDA and kidney MDA. In Study 2, liver iron and breath ethane decreased progressively (P < 0.05) from 1 d to 3 d after the last iron dose in intermittently supplemented rats. We conclude that iron deficiency results in lipid peroxidation, but that its correction with daily iron supplements results in abnormal iron accumulation and increased lipid peroxidation in rats. These effects are mitigated by intermittent iron supplementation.
Assuntos
Dieta , Deficiências de Ferro , Ferro/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Testes Respiratórios , Relação Dose-Resposta a Droga , Esquema de Medicação , Etano/química , Cromatografia Gasosa-Espectrometria de Massas , Ferro/administração & dosagem , Rim/metabolismo , Fígado/metabolismo , Masculino , Malondialdeído/sangue , Malondialdeído/química , Estado Nutricional , Ratos , Ratos Sprague-Dawley , Vitamina E/sangueRESUMO
Oxidative DNA damage is important in aging and the degenerative diseases of aging such as cancer. Estimates commonly rely on measurements of 8-oxo-2'-deoxyguanosine (oxo8dG), an adduct that occurs in DNA and is also excreted in urine after DNA repair. Here we examine difficulties inherent in the analysis of oxo8dG, identify sources of artifacts, and provide solutions to some of the common methodological problems. A frequent criticism has been that phenol in DNA extraction solutions artificially increases the measured level of oxo8dG. We found that phenol extraction of DNA contributes a real but minor increase in the level of oxo8dG when compared, under equivalent conditions, with a successful nonphenol method. A more significant reduction in the baseline level was achieved with a modification of the recently introduced chaotropic NaI method, reducing our estimate of the level of steady-state oxidative adducts by an order of magnitude to 24,000 adducts per cell in young rats and 66,000 adducts per cell in old rats. Of several alternative methods tested, the use of this chaotropic technique of DNA isolation by using NaI produced the lowest and least variable oxo8dG values. In further studies we show that human urinary 8-oxo-guanine (oxo8Gua) excretion is not affected by the administration of allopurinol, suggesting that, unlike some methylated adducts, oxo8Gua is not derived enzymatically from xanthine oxidase. Lastly, we discuss remaining uncertainties inherent both in steady-state oxo8dG measurements and in estimates of endogenous oxidation ("hit rates") based on urinary excretion of oxo8dG and oxo8Gua.