RESUMO
AIMS: Fructose (F) overload in rats induces metabolic dysfunctions that resemble the human metabolic syndrome. In this paper, we aimed to investigate the response of F overload rats to lipopolysaccharide (LPS) challenge in terms of nitric oxide (NO) production and prostanoids (PR) release. MAIN METHODS: NO blood steady-state concentration was monitored through the detection of nitrosyl-hemoglobin complexes (NO-Hb) by electronic spin resonance. Production of 6-keto PGF(1)α, PGE(2), PGF(2)α and TXB(2) was measured in aorta and mesenteric beds by HPLC. Western blot analysis was used to examine the changes in the expression levels of NOS-2 and COX-2 in aorta. KEY FINDINGS: Our results showed that increases in NO circulating steady-state concentration and PR production by aorta and mesenteric beds 6h after LPS administration were significantly attenuated in F overload rats with respect to control animals. Oxidative stress parameters were equally affected in the presence or absence of the F treatment. Aorta protein levels of NOS-2 and COX-2, two enzymes inducible by LPS, were significantly lower in F overload rats with respect to control rats at the end of the treatment (-39% and -61% for NOS-2 and COX-2 respectively). SIGNIFICANCE: These results suggest that the metabolic alterations established by 15 weeks of F overload should affect the response to LPS challenge due to an attenuation in the induction of NOS-2 and COX-2. This effect would be one of the components contributing to abnormalities in the course of the inflammatory response in other conditions associated to insulin resistance, such as diabetes.
Assuntos
Ciclo-Oxigenase 2/biossíntese , Frutose/farmacologia , Lipopolissacarídeos/farmacologia , Óxido Nítrico Sintase Tipo II/biossíntese , Animais , Glicemia/análise , Western Blotting , Ciclo-Oxigenase 2/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Insulina/sangue , Masculino , Nitratos/sangue , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Nitritos/sangue , Estresse Oxidativo/efeitos dos fármacos , Prostaglandinas/metabolismo , Ratos , Ratos Sprague-Dawley , Triglicerídeos/sangueRESUMO
Iron is essential for human life, however it can be toxic through Fe ability to generate oxygen-derived radicals. This work reviews the main features of Fe binding in the cell and its association to these processes. The chemical nature of the Fe extracted by chelation therapy in pathophysiological situations is also analyzed.
Assuntos
Terapia por Quelação , Quelantes de Ferro/química , Quelantes de Ferro/uso terapêutico , Ferro/metabolismo , Animais , HumanosRESUMO
The ability of the ESR technique based on diethyldithiocarbamate (DETC) administration was studied as a suitable method to assess NO generation in vivo. The technique was successfully employed to measure NO generation after LPS treatment. DETC2-Fe-NO adducts were detected in liver homogenates of iron overloaded animals. When iron was administered to the animals simultaneously with LPS, NO-dependent signal increased 122%, but the content of NO2- and NO3- in sera was significantly lower (44%) as compared to LPS-treated rats. Iron dextran administration was responsible for a three-fold increase in the DETC2-Fe-NO content in non-LPS treated rats, while NOS activity and sera NO2- and NO3- levels remained unaffected. The adduct generation rate by a chemical NO-source was recorded in the presence of either control or iron overloaded homogenates supplemented with DETC in vivo. The exposure of liver homogenates to NO was performed either by the addition of 1 mM SNAP as NO donor or infusing an aqueous NO solution. In the presence of iron overloaded samples the adduct generation rate was 3.8-4.4-fold higher than in the presence of control samples. This effect restricts the applicability of the method to experimental conditions where iron levels remain constant, therefore it is not suitable for NO generation studies in experimental models where animals were subjected to iron overload.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Sobrecarga de Ferro , Óxido Nítrico/análise , Penicilamina/análogos & derivados , Animais , Ditiocarb/metabolismo , Ditiocarb/farmacologia , Interações Medicamentosas , Escherichia coli/imunologia , Técnicas In Vitro , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Nitratos/sangue , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Nitritos/sangue , Penicilamina/farmacologia , Ratos , Ratos Wistar , Detecção de SpinRESUMO
The involvement of cytosolic nitric oxide (NO) and mitochondrial superoxide radical (O2(.-)) production was evaluated as a mechanism triggering liver oxidative stress in lindane (40 mg/kg) or L-3,3',5-triiodothyronine (T3, 0.1 mg/kg for 2 consecutive days) treated animals (male Sprague-Dawley rats) subjected to iron overload (200 mg/kg). Lindane and iron led to 504 and 210% increases in the content of hepatic protein carbonyls as an index of oxidative stress, with a 706% enhancement being produced by their combined administration. T3 did not alter this parameter, whereas iron overload increased the content of protein carbonyls by 116% in hyperthyroid rats. Lindane increased NO generation by 106% without changes in generation of O2(.-), whereas iron enhanced both parameters by 109 and 80% over control values, respectively, with a net 33 and 46% decrease, respectively, being elicited by the combined treatment related to iron overload alone. Hyperthyroidism increased liver NO (69%) and O2(.-) (110%) generation compared to controls, effects that were either synergistically augmented or suppressed by iron overload, respectively. The in vitro addition of iron (1 micromol/mg protein) to liver cytosolic fractions from euthyroid (97%) and hyperthyroid (173%) rats also enhanced NO generation. The effects of iron overload on mitochondrial O2(.-) production by hyperthyroid rats were reproduced by the in vitro addition of 1 micromol iron/mg protein and abolished by the in vivo pretreatment with the iron chelator desferrioxamine (500 mg/kg). It is concluded that liver oxidative stress induced by iron overload is independent of NO and O2(.-) production in lindane-treated rats, whereas in hyperthyroid animals NO generation is a major factor contributing to this redox imbalance.
Assuntos
Hexaclorocicloexano/toxicidade , Ferro/farmacologia , Fígado/efeitos dos fármacos , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Hormônios Tireóideos/toxicidade , Animais , Fígado/metabolismo , Masculino , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Parameters related to liver oxidative stress, Kupffer cell function, and hepatocellular injury were assessed in control rats and in animals subjected to lindane (40 mg/kg; 24 h) and/or iron (200 mg/kg; 4 h) administration. Independently of lindane treatment, iron overload enhanced the levels of iron in serum and liver. Biliary efflux of glutathione disulfide increased by 140, 160, or 335% by lindane, iron, or their combined administration, respectively, and the hepatic content of protein carbonyls was elevated by 5.84-, 2.95-, and 10-fold. Colloidal carbon uptake by perfused livers was not modified by lindane and/or iron, whereas gadolinium chloride (GdCl(3)) pretreatment diminished uptake by 60-72%. Carbon-induced liver O(2) uptake was not altered by lindane, whereas iron produced a 61% increase and the combined treatment led to a 72% decrease over control values. Pretreatment with GdCl(3) abolished these effects in all groups. Lindane-treated rats showed acidophilic hepatocytes in periportal areas and some hepatic cells with nuclear pyknosis, whereas iron overload led to moderate hyperplasia and hypertrophy of Kupffer cells and moderate inflammatory cell infiltration. Combined lindane-iron treatment led to hepatocytes with pyknotic nuclei, significant acidophilia, and extensive lymphatic and neutrophil infiltration in the portal space. Hepatic myeloperoxidase activity increased by 1.1-, 2.1-, or 6.7-fold by lindane, iron, or their combined administration, respectively. Liver sinusoidal lactate dehydrogenase efflux increased by 2.2-fold (basal conditions) and 9.7-fold (carbon infusion) in the lindane-iron treated rats, effects that were diminished by 35 and 78% by GdCl(3) pretreatment, respectively. These data support the contention that lindane sensitizes the liver to the damaging effects of iron overload by providing an added enhancement to the oxidative stress status in the tissue, and this may contribute to the alteration of the respiratory activity of Kupffer cells and the development of an inflammatory response.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Hexaclorocicloexano/toxicidade , Inseticidas/toxicidade , Sobrecarga de Ferro/patologia , Células de Kupffer/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Animais , Carbono/farmacologia , Ferro/metabolismo , Ferro/farmacocinética , Sobrecarga de Ferro/metabolismo , L-Lactato Desidrogenase/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Peroxidase/metabolismo , Fagocitose/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
The effect of dietary alpha-tocopherol (alpha-T) supplementation on iron overload-dependent oxidative damage was studied. Male Wistar rats were fed diets supplemented with 2.5% carbonyl iron and/or 200 mg/kg of alpha-tocopheryl acetate for 6 weeks. Oxidation of lipids and proteins were increased by iron overload in rat liver, and alpha-T dietary supplementation effectively prevented these effects. Iron overload decreased both, catalase and Mn-superoxide dismutase activities by 49 and 54%, respectively, with no effect on glutathione peroxidase activity. Alpha-T supplementation did not prevent the inhibition measured in catalase and Mn-superoxide dismutase activities. Iron dietary excess had no effect on liver alpha-T and ubiquinol 9 (UQ9) content. Ubiquinol 10 (UQ10) content after iron overload was decreased by 58 and 54% in whole liver and liver mitochondria, respectively. Alpha-T supplementation led to significant increases in alpha-T, UQ9 and UQ10 content in liver, as compared to control values, and partially prevented the decrease in UQ10 content due to iron excess. The results presented here indicate that initial stages of iron overload led to oxidative damage in liver (evaluated in terms of lipid and protein oxidation) with a decline in antioxidant defenses. alpha-T supplementation affected the liver content of lipid soluble antioxidants, suggesting a concerted antioxidant response at the cellular level to modulate the effect of excess iron availability.
Assuntos
Antioxidantes/farmacologia , Sobrecarga de Ferro/metabolismo , Vitamina E/farmacologia , Animais , Catalase/metabolismo , Peroxidação de Lipídeos , Masculino , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/análiseRESUMO
Iron overload was developed in rats by ip injection of iron-dextran. Iron concentration in plasma increased 12-fold after 20 h of iron supplementation and unsaturated iron binding capacity (UIBC) drastically decreased in iron overloaded compared to control rats (69 +/- 36 and 177 +/- 19 micrograms/dl, respectively). Lipid peroxidation in plasma increased by 285% and plasma alpha-tocopherol content decreased by 40% after 20 h of iron overload. alpha-Tocopherol supplementation decreased by 30% the measured increase in TBARS content in plasma after iron injection. On the other hand, both iron and TBARS content in erythrocytes were not significantly different in control and iron loaded rats. However, red blood cells from iron treated rats exposed to pro-oxidant conditions showed a significant increase in TBARS content as compared to control erythrocytes. alpha-Tocopherol pretreatment prevented this increase. Moreover, red blood cells from iron loaded rats showed a higher content of TBARS after incubation with plasma from iron-dextran injected rats than after incubation with plasma from control animals. This measured increase was partially prevented by alpha-tocopherol supplementation. Neither the activity of antioxidant enzymes nor the content of alpha-tocopherol in red blood cells were affected by iron overload. Total thiols content was significantly lower (30%) in erythrocytes isolated from iron treated rats. The data presented here suggest that free radical generation catalyzed by metal ions may lead to consumption of thiols. The decrease in thiols content in erythrocytes could afford an appropriate degree of protection and avoid other oxidative damage to these cells.
Assuntos
Antioxidantes/farmacologia , Eritrócitos/efeitos dos fármacos , Complexo Ferro-Dextran/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Animais , Eritrócitos/química , Ferro/análise , Ferro/sangue , Masculino , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Vitamina E/sangue , Vitamina E/farmacologiaRESUMO
A single injection of iron-dextran significantly increased iron content in plasma, whole liver, cellular cytosol and liver nuclei. In vitro nuclear rate of Fe(3+)-EDTA reduction was not affected by the treatment. Membrane-bound enzymatic activities in the nuclei were measured after iron overload. Both NADPH- and NADH-dependent cytochrome c reductases were slightly decreased after iron overload, but cytochrome P450 was undetectable after 6 h of iron supplementation. The contents of lipid- and water-soluble antioxidants were measured in isolated nuclei from control and iron-overloaded rats. alpha-Tocopherol and beta-carotene co-elutant were decreased by 40% and 83%, respectively after 6 h of treatment. Nuclear glutathione content was not affected. The rate of generation of superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical-like species by isolated rat liver nuclei, were decreased by 50%, 40% and 60%, respectively after 6 h of iron supplementation. An identical qualitative response to iron overload was observed with NADPH and NADH. The inactivation of nuclear cytochrome P450, the significant loss in lipid-soluble antioxidants (alpha-tocopherol and beta-carotene) and the decrease in enzyme-dependent oxygen radical generation, suggest that the increase in catalytic active iron induced by iron overload could affect the cellular nuclei functionality.
Assuntos
Núcleo Celular/efeitos dos fármacos , Ferro/toxicidade , Fígado/efeitos dos fármacos , Animais , Antioxidantes/análise , Núcleo Celular/metabolismo , Radical Hidroxila , Complexo Ferro-Dextran/administração & dosagem , Fígado/metabolismo , Masculino , NADH Desidrogenase/metabolismo , Ratos , Ratos Wistar , Superóxidos/metabolismoRESUMO
1. Hepatotoxicity is the most common finding in patients with iron overload since the liver is the major recipient of iron excess, even though the kidney could be a target of iron toxicity. The effect of iron overload was studied in the early stages after iron-dextran injection in rats, as a model for secondary hemocromatosis. 2. Total hepatic and kidney iron content was markedly elevated over control values 20 h after the iron administration. Plasma GOT, GPT and LDH activities were not affected, suggesting that liver cell permeability was not affected by necrosis. 3. Spontaneous liver chemiluminescence was measured as an indicator of oxidative stress and lipid peroxidation. Light emission was increased four-fold 6 h after iron supplementation. 4. Increases in the generation of thiobarbituric acid reactive substances (TBARS in liver and kidney homogenates were detected after iron administration. 5. The activities of catalase, SOD and glutathione peroxidase were determined. Enzymatic activities declined in liver homogenates by 25, 36 and 32%, respectively, 20 h after iron injection. These activities were not affected in kidney as compared to control values, except for SOD activity that was decreased by 26%. 6. The content of alpha-tocopherol was decreased by 31% in whole kidney homogenates and by 40% in plasma. 7. Our data indicate that lipid peroxidation occurs after mild iron overload both in liver and kidney. Enzymatic antioxidants are consumed significantly in liver and alpha-tocopherol content decreases in kidney, suggesting an organ-specific antioxidant effect.
Assuntos
Ferro/administração & dosagem , Rim/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Animais , Permeabilidade da Membrana Celular , Rim/química , Fígado/química , Medições Luminescentes , Masculino , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico , Vitamina E/análiseRESUMO
1. Hepatotoxicity is the most common finding in patients with iron overload since the liver is the major recipient of iron excess, even though the kidney could be a target of iron toxicity. The effect of iron overload was studied in the early stages after iron-dextran injection inrats, as a model for secondary hemocromatosis. 2. Total hepatic and kidney iron content was markedly over control values 20h after the iron administration. Plasma GOT, GPT and LDH activities were not affected, suggesting that liver cell permeability was not affected by necrosis. 3. Spontaneous liver chemiluminescence was measured as an indicator of oxidative stress and lipid peroxidation. Light emission was increased four-fold 6 h after iron supplementation. 4. Increases in the generation of thiobarbituric acid reactive substances (TBARS) in liver and kidney homogenates were detected after iron administration. 5. The activities of catalase, SOD and glutathione peroxidase were determined. Enzymatic activities declined in liver homogenates by 25, 36 and 32 por cento, respectively, 20 h after iron injection. These activities were not affected in kidney as compared to control values, except for SOD activity that was decreased by 26 por cento. 6. The content of alfa-tocopherol was decreased by 31 por cento in whole kidney homogenates and by 40 por cento in plasma. 7. Our data indicate that lipid peroxidation occurs after mild iron overload both in liver and kidney. Enzymatic antioxidantes are consumed significantly in liver and alfa-tocopherol content decreases in kidney, suggesting an organ-specific antioxidant effect
Assuntos
Animais , Masculino , Ratos , Fígado/metabolismo , Ferro/administração & dosagem , Rim/metabolismo , Permeabilidade da Membrana Celular , Fígado/química , Ferro/toxicidade , Rim/química , Medições Luminescentes , Peroxidação de Lipídeos , Ratos Wistar , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico , Vitamina E/químicaRESUMO
The effect of iron-overload on rat kidney was studied after a single injection of iron-dextran. Total iron content in kidney and isolated kidney mitochondria was markedly elevated over control values. To assess mitochondrial damage by iron overload, succinate-cytochrome c reductase and NADH-cytochrome c reductase activities as well as the rate of succinate-dependent hydrogen peroxide generation were measured. None of these activities were significantly affected by acute iron overload. The net content and the rate of TBARS (thiobarbituric acid reactive species) formation in kidney homogenates from iron-treated rats was significantly higher than that of control animals. Total superoxide dismutase activity in the homogenates from iron overloaded kidney was decreased by 26%, as compared to controls. Catalase, glutathione peroxidase, and Mn-superoxide dismutase activities were not affected by the treatment. The content of alpha-tocopherol was consistently decreased in whole kidney homogenates (-31%), mitochondria from kidney medulla (-31%) and cortex (-34%), from iron-overloaded rats. Our data suggest that iron dextran treatment does not affect kidney integrity, even though increases in lipid peroxidation occur. Vitamin E appears to be effective in controlling iron-dextran dependent radical generation in kidney.
Assuntos
Membranas Intracelulares/efeitos dos fármacos , Ferro/toxicidade , Rim/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Injeções Intraperitoneais , Membranas Intracelulares/metabolismo , Ferro/administração & dosagem , Ferro/análise , Rim/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Oxirredução , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Vitamina E/metabolismoRESUMO
The effect of iron-overload on both hepatic lipid peroxidation and chemiluminescence was studied in early stages after iron-dextran injection. Total hepatic iron content was markedly elevated over control values 2-6 h after iron dose. A 4-fold increase in light emission was detected after 4-6 h after iron injection. Plasma GOT, GPT and LDH activities were not affected by the treatment suggesting that cell permeability was not affected by necrosis. Increases in the generation of thiobarbituric acid reactive substances (TBARS) and chemiluminescence in liver homogenates, were determined as a function of time after iron administration, in the presence of NADPH as cofactor. Under the same experimental conditions, microsomal cytochrome P-450 content was decreased by 40%, 2 h after iron treatment. To evaluate liver antioxidant defenses, catalase, superoxide dismutase and glutathione peroxidase activities were determined. Glutathione peroxidase activity in the homogenate was not affected by the treatment. Catalase and superoxide dismutase activities declined by 25 and 36%, respectively, compared with control values 4 h after the iron dose. Our data suggest that lipid peroxidation occurs after mild iron overload even though the liver remains functional.
Assuntos
Ferro/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Animais , Fígado/metabolismo , Medições Luminescentes , Masculino , Ratos , Ratos WistarRESUMO
The total rate of mitochondrial O2- production in the presence of NADH as substrate increased from 200 to 1340 pmol/min per axis between 2 and 30 h of imbibition. The activities of the enzymes involved in hydroperoxide metabolism, e.g., superoxide dismutase, catalase, peroxidase and glutathione and ascorbate peroxidases, markedly changed during the germination of soybean embryonic axes. Superoxide dismutase was the enzymatic activity affected the most during the initial stages of germination. Intracellular O2- steady-state concentration, calculated from the rate of O2- production and superoxide dismutase activity, showed a 2-fold increase from 2 x 10(-8) M to 4 x 10(-8) M in germination phase I, declined in phase II to 2 x 10(-8) M and remained constant over the rest of the incubation period. The reaction of H2O2 and luminol catalyzed by Co2+ was utilized to measure H2O2 diffused out of the soybean axes after 5 to 10 min of incubation. The catalase-sensitive luminol emission of diffusates prepared from axes previously imbibed from 2 to 30 h corresponded to a H2O2 intracellular steady-state concentration in the range of 0.3 to 0.9 microM. The activity of metal-containing antioxidant enzymes was determined in the extracellular fluid. Cell wall peroxidase activity increased from 10 to 300 mumol/min per mg protein and appears as a potentially important pathway for H2O2 utilization. Hydrogen peroxide metabolism in soybean embryonic axes during early inhibition appears to have the following main features: (a) mitochondrial membranes are the most important source of cytosolic O2- and H2O2; (b) H2O2 is regulated at a steady-state concentration of 0.3-0.9 microM; (c) catalase is the main enzyme in terms of H2O2 utilization; (d) H2O2 exo-diffusion is quantitatively important destiny of intracellular H2O2; and (e) extracellular peroxidase located at the cell wall affords an enzymatic system able to use diffused H2O2.
Assuntos
Glycine max/embriologia , Peróxido de Hidrogênio/metabolismo , Sementes/metabolismo , Superóxidos/metabolismo , Catalase/metabolismo , Espaço Extracelular/metabolismo , Glutationa Peroxidase/metabolismo , Medições Luminescentes , Mitocôndrias/metabolismo , Consumo de Oxigênio , Superóxido Dismutase/metabolismoRESUMO
Human breast tumor cells MCF-7 were grown during 5 days in the presence of Adriamycin and the IC50 was 50 nM with the highest sublethal concentration 0.1 microM. At this latter concentration Adriamycin produced a complete inhibition of cell division and a partial reversion to a normal breast epithelial appearance. Similar effects of Adriamycin were observed in cells cultured in the presence of 10% FBS and in a chemically defined medium, with Se-glutathione peroxidase activities of 3.8 and 1.3 U/mg of protein, respectively. Cell size and cell oxygen uptake were increased by 41% and by 50%, respectively, in Adriamycin-treated cells. The spontaneous chemiluminescence of monolayers of intact MCF-7 cells (81 +/- 9 cps/mg protein) was increased by 48% in the Adriamycin-treated cultures (120 +/- 11 cps/mg of protein) in agreement with a 91% higher concentration of malondialdehyde in the same cultures. Adriamycin treatment produced a 71% increase in the steady state concentration of H2O2, which was estimated assuming diffusion equilibrium with the external medium, from 1.38 microM in the control cells to 2.38 microM in the treated cells. Cyanide-insensitive respiration was also higher in the cells exposed to the drug than in the control cells. Adriamycin did not affect the activity of the antioxidant enzymes, Cu-Zn and Mn-superoxide dismutase, Se and non-Se-glutathione peroxidase, and catalase. These results contribute to the current hypothesis that oxygen free radicals produced by Adriamycin redox cycling are responsible for at least part of the cytotoxic effects due to this drug.