RESUMO
Neutrophils, eosinophils and macrophages interact with invading parasites and naive hosts. The initial reaction of leukocytes is the generation of reactive oxygen species (ROS). The cytotoxic effects of extracts derived from intact Cysticercus cellulosae and from the scolex or membrane fractions on neutrophils were examined. DNA fragmentation of neutrophils was observed when cells were incubated with an extract from the intact metacestode; however, the addition of antioxidant enzymes to the incubation medium had a protective effect. The scolex and membrane extracts did not affect DNA fragmentation of neutrophils. Hydrogen peroxide production of neutrophils incubated with metacestode fractions from C. cellulosae increased by 190% (total extract), 120% (scolex) or 44% (membrane). An increase in antioxidant catalase activity (28%) concomitant with the increased production of ROS was observed in neutrophils incubated with metacestode fractions, which could be an attempt at self-protection. ROS production by neutrophils in the presence of the intact cysticerci extract did not alter phagocytosis. In contrast, the scolex and membrane fractions increased the phagocytic capacity of neutrophils by 44 and 28%, respectively. The results showed that the extract from intact C. cellulosae was toxic for neutrophils via ROS production, leading to DNA fragmentation and inhibition of phagocytic capacity, but neutrophils are able to protect themselves against oxidative stress by via catalase activity.
Assuntos
Cysticercus/imunologia , Fragmentação do DNA , DNA de Helmintos/imunologia , Neutrófilos/fisiologia , Neutrófilos/parasitologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Catalase/metabolismo , Células Cultivadas , Cisticercose/parasitologia , Cisticercose/veterinária , Cysticercus/enzimologia , Cysticercus/genética , Citometria de Fluxo/veterinária , Glutationa Peroxidase/metabolismo , Peróxido de Hidrogênio/metabolismo , Masculino , Neutrófilos/enzimologia , Estresse Oxidativo , Fagocitose , Superóxido Dismutase/metabolismo , Suínos , Doenças dos Suínos/parasitologiaRESUMO
1. The effect of fish oil (FO) administration by gavage (0.4% body weight) on macrophage and lymphocyte function was investigated in young male rats. The results were compared with those obtained by administration of soybean oil (SB) and cocoa butter (CB). 2. Lymphocyte proliferation was markedly increased by FO administration compared with control and other oils. 3. Macrophage phagocytosis capacity was not affected by FO, but it was increased by CB and SB. 4. The oils did not affect the production of O2.- but increased the production of H2O2 in the presence of PMA. 5. The administration of the oils did not markedly affect the activity of antioxidant enzymes in macrophages, except for a decrease in superoxide dismutase activity by FO.
Assuntos
Óleos de Peixe/farmacologia , Peróxido de Hidrogênio/metabolismo , Linfócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Animais , Catalase/sangue , Divisão Celular/efeitos dos fármacos , Gorduras na Dieta/farmacologia , Glutationa Peroxidase/sangue , Intubação Gastrointestinal , Linfócitos/citologia , Masculino , Ratos , Óleo de Soja/farmacologia , Superóxido Dismutase/sangueRESUMO
Changes in the integrity, ultrastructure, phagocytosis capacity, and production of H2O2, O2.- and NO2- were evaluated in cultured neutrophils. The activities of the antioxidant enzymes (catalase-CAT, superoxide dismutase-SOD and glutathione-dependent peroxidase-GSH-Px) were measured under similar conditions. The integrity of the cells remained unchanged up to 18 h. After 24 h, the number of viable cells in culture dropped by 16 per cent. The percentage of viable cells in culture was of 72 per cent even after 72 h. An ultrastructural analysis of the cells was carried out after 3, 6, 12, 24, 48, and 72 h in culture. Neutrophils started developing morphologic changes after 24 h: decreased cell volume, abundant vacuoles (mainly around the nucleus), and also the presence of autophagic vacuoles. This period was then chosen for the study of neutrophil function and antioxidant enzyme activities. Neutrophils cultured for 24 h presented reduced phagocytosis capacity. The rates of production of H2O2 and O2.- remained unchanged after 24 h in culture. Concomitantly, these cells were also able to produce NO in significant amounts. The production of O2.- in response to PMA stimulus was lowered in 24-h cultured cells. Possibly, the production of oxygen and nitrogen reactive species accomplished with a decrease in the activities of CAT and GSH-Px play a key role for the process of apoptosis which takes place in neutrophils under these conditions.
Assuntos
Antioxidantes/metabolismo , Neutrófilos/fisiologia , Fagocitose , Animais , Catalase/metabolismo , Células Cultivadas , Glutationa Peroxidase/metabolismo , Peróxido de Hidrogênio/metabolismo , Masculino , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Nitritos/metabolismo , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo , Superóxidos/metabolismoRESUMO
The capacity of rat neutrophils to utilize glutamine was investigated by 1) determination of oxygen consumption in the presence of glucose or glutamine, 2) measurement of maximal activity of phosphate-dependent glutaminase, 3) Northern blot, Western blot, and immunocytochemical detection of glutaminase, and 4) measurement of glutamine utilization and also production of ammonia, glutamate, aspartate, alanine, and lactate and decarboxylation of [U-14C]glutamine in cells incubated for 1 h. The rate of respiration by isolated neutrophils in the absence of added substrate was 5.0 nmol x min(-1) x 10(7) cells(-1). Maximal activity of phosphate-dependent glutaminase was 56 nmol x min(-1) x mg protein(-1) in freshly obtained neutrophils; the Michaelis-Menten constant was 3.5 mM for glutamine. This enzyme activity was inhibited by 2 mM glutamate, 2 mM oxoglutarate, and 2 mM NH4Cl. The presence of glutaminase protein (65 kDa) was confirmed by Western blot and immunocytochemical detection and the presence of the mRNA (6.0 kb) by Northern blot analysis. Glutamine was utilized by neutrophils incubated for 1 h at a rate of 12.8 nmol x min(-1) x mg protein(-1) when the amino acid was added to the medium at 2 mM, which is three to four times higher than the physiological concentration. In the presence of 0.5 mM glutamine, the amino acid was utilized at a rate of 2.9 nmol x min(-1) x mg protein(-1). The addition of 0.5 mM glutamate to the incubation medium caused a marked reduction (by 70%) in glutamine utilization by neutrophils. Glucose was utilized at 7.7 nmol x min(-1) x mg protein(-1) when cells were incubated in 5 mM glucose. The conversion of [U-14C]glutamine to 14CO2 was very low: <1% was totally oxidized. The formation of ammonia was approximately 27% of glutamine utilization, and the conversion of glutamine to glutamate, aspartate, alanine, and lactate accounted for approximately 84.6% of the total amino acid utilized by neutrophils. In this study, evidence is presented that, in addition to lymphocytes and macrophages, neutrophils also utilize glutamine.
Assuntos
Glutaminase/sangue , Glutamina/sangue , Neutrófilos/metabolismo , Animais , Glicemia/metabolismo , Western Blotting , Glutaminase/biossíntese , Técnicas In Vitro , Cinética , Masculino , Consumo de Oxigênio , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Transcrição GênicaRESUMO
Peroxidase activity in neutrophils is higher than in thioglycollate macrophages, while in lymphocytes this enzyme activity is very low. Indole-3-acetic acid is oxidized by peroxidase and the role of this enzyme in the cytotoxic effect of the compound was evaluated by measuring oxygen consumption, light emission and cell death in neutrophils, macrophages and lymphocytes. The increase in light emission, oxygen consumption and rate of cell death in cells cultured in the presence of indole-3-acetic acid presented a direct correlation with the peroxidase activity of the cells as follows: neutrophils > thioglycollate macrophages > resident macrophages > lymphocytes. Indeed, in lymphocytes that possess very low peroxidase activity, indole-3-acetic acid did not result in an increase in light emission or oxygen consumption and it was not cytotoxic.
Assuntos
Ácidos Indolacéticos/toxicidade , Linfócitos/enzimologia , Macrófagos/enzimologia , Neutrófilos/enzimologia , Peroxidase/metabolismo , Reguladores de Crescimento de Plantas/toxicidade , Animais , Células Cultivadas , Linfócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
The peroxidative metabolism of N-methylcarbazole emits light independently of the presence of oxygen. It is likely that two chemiexcited transients are formed by electron transfer to the activated peroxidase, the cation radical by one electron transfer and a cation biradical by two electron transfer consistent with the failure to observe horseradish peroxidase-II in the steady state of the reaction. In the spectral range investigated (390-700 nm) the observed emission (570-700 nm) is ascribed to the biradical, as the latter is equivalent to an excited state of the postulated iminium cation. While lipoxygenase has no effect upon N-methylcarbazole, it markedly enhances the emission if peroxidase is present. This effect requires oxygen and is ascribed to an excited product formed by lipoxygenase acting upon an intermediate hydroperoxide of the aerobic process promoted by peroxidase. Our results are of importance on two counts. First they extend to N-methylcarbazole the formation of excited species in the peroxidative metabolism of important xenobiotics. Second, the mechanistic information they provide supports the scheme of metabolism postulated by Kedderis et al. (1986, J. Biol. Chem. 261, 15910-15914).
Assuntos
Carbazóis/metabolismo , Carbazóis/química , Transporte de Elétrons , Peroxidase do Rábano Silvestre/metabolismo , Técnicas In Vitro , Luz , Lipoxigenase/metabolismo , FotoquímicaRESUMO
Light emission from the horseradish peroxidase-catalyzed aerobic or anaerobic oxidation of indole-3-acetic acid has been investigated under opposite extreme conditions of enzyme/substrate ratio. The O2-dependent chemiluminescent processes represent a minor part of the total oxygen consumption. Superoxide is involved in chemiexcitation as is evident from the observed inhibitory effect of superoxide dismutase. At high enzyme/substrate ratio, only a part of the emission is dependent on superoxide ion; at low ratio the dependence is extensive. At high ratio, some of the emission is independent of superoxide and O2. The identical quenching effects of D- and L-tryptophan are consistent with the formation of the quenching species only in bulk solution. The similarity of the emission spectra under extreme conditions indicates that the same main emitters are formed. This is also supported by the effect of quenchers. Possibly some of the emitters originate in the oxidative cleavage of the 2,3-double bond of the indole ring.