Intracellular Antioxidant Enzyme Activities Research Articles

Does the platelet-activating factor affect the antioxidant defense system? The possible role of hyperbaric oxygenation.

The platelet-activating factor (PAF) is an inflammatory mediator and it may exert some of its effects by reactive oxygen species (ROS). We investigated the effects of PAF and hyperbaric oxygenation (HBO) on copper (Cu) and zinc (Zn) levels in plasma and the intracellular antioxidant enzyme activities of rats. PAF administration caused a decrease in erythrocyte catalase (CAT) and glutathione peroxidase (GPx) activities and in the plasma zinc level. Following PAF administration, exposure to HBO also caused a decrease in erythrocyte GPx activity. These results support the hypothesis that PAF may produce free oxygen radicals and HBO enhances this effect. The enzyme activities of the antioxidant defense system were found to be affected by these oxidative processes. This is likely to be the result of excessive production of ROS or overutilization and/or inhibition of the antioxidant enzymes.

Effect of Dissolved Oxygen Concentration on Superoxide Dismutase Production by Humicola lutea Cells

Cultures of the fungal strain Humicola lutea 110 were grown in a 3-l bioreactor. Effects of dissolved oxygen concentration (DO) on cell growth, intracellular protein content and antioxidant enzyme activities (SOD and catalase) were investigated. Controlling DO from 20 to 60% lead to: (I). The lethal phase of growth was reached faster; (ii) strong reduction of the intracellular protein content, and (iii) increase of antioxidant enzyme activities. The most efficient SOD biosynthesis was achieved at the 1st maximum of activity in the culture grown under DO uncontrolled conditions.

L-arginine increases UVA cytotoxicity in irradiated human keratinocyte cell line: potential role of nitric oxide.

Human fibroblasts and keratinocytes possess nitric oxide synthases (NOS), which metabolize L-arginine (L-Arg) for producing nitric oxide (NO*). This report delineates the relations between NO* and UVA in the human keratinocyte cell line HaCaT. NOS activity was stimulated by exposure of cells to L-Arg just after irradiation. L-Arg (5 mM) supply led to an increase in UVA (25.3 J/cm(2)) cytotoxicity (% of viability 18 +/- 3%) whereas neither L-Arg itself nor UVA irradiation induced cell death at the doses used in this study. Cells were also treated either with L-thiocitrulline (L-Thio), an irreversible inhibitor of NOS, or with exogenous superoxide dismutase (SOD) and catalase. L-Thio and SOD prevented L-Arg-mediated deleterious effects in irradiated cells, whereas catalase was ineffective. Intracellular antioxidant enzyme activities were also determined. UVA/L-Arg stress altered catalase (66% decrease) and glutathione peroxidase (83% decrease). DNA damage was evaluated using the 'comet assay' and quantified using the 'tail moment'. UVA alone was genotoxic (mean tail moment: 25.43 +/- 1.23, P<0.001 compared control cells). The addition of L-Arg potentiated DNA damage (mean tail moment: 41.05+/-3.9) whereas L-Thio prevented them (mean tail moment 9.86 +/- 0.98). We attempted to assess the effect of poly(ADP-ribose) polymerase (PARP) inhibition on cell death. Using the PARP inhibitor 3-aminobenzamide, we established that PARP determines both cell lysis and DNA damage induced by UVA and/or L-Arg. Our findings demonstrated that L-Arg was able to increase UVA-mediated deleterious effects in keratinocytes (both DNA damage and cytotoxicity) and that the ratio NO*/O2*- plays a key role in these processes.

Alterations in superoxide dismutase, glutathione peroxidase and catalase activities in experimental cerebral ischemia-reperfusion.

Free radicals are thought to be the most important cause of the reperfusion injury subsequent to ischemia. The antioxidant status of the tissue affected by ischemia-reperfusion is of great importance for the primary endogenous defense against the free radical induced injury. This investigation was performed to evaluate the antioxidant enzyme capacity of the brain tissue in the ischemia-reperfusion period using an experimental global moderate (penumbral) ischemia model on rat brains. Experiments were performed on 45 male Sprague Dawley rats. Ischemia was induced by bilateral vertebral arteries cauterization and temporary bilateral carotid arteries occlusion and sustained for 10 minutes. At the end of ischemia (0 min reperfusion) and various reperfusion periods (20 min, 60 min, 240 min), rats were decapitated and brains were frozen in liquid nitrogen. Changes in the intracellular antioxidant enzyme (superoxide dismutase, glutathione peroxidase and catalase) activities were assessed in the rat brain tissues, by spectrophotometric methods. In all moderate ischemia-reperfusion groups, superoxide dismutase activities were found to have decreased significantly compared to the sham operated controls (P < 0.05). During ischemia superoxide dismutase activity was lowered to 31% of that of the control group. The decreases were more significant in reperfusion groups, particularly in 60 min reperfusion (40%). Relatively smaller but still significant diminution was observed in glutathione peroxidase activities (P < 0.05). The ratio of diminution was striking in 20 min and 60 min reperfusion groups with 26% of the sham operated rats. Conversely, moderate ischemia-reperfusion caused significant increase in catalase activities (P < 0.05). The increment was 63% of the preischemic level with 10 min of moderate ischemia. In conclusion, activities of the major antioxidant enzymes were changed significantly in moderate brain ischemia-reperfusion. These results suggest that the disturbance in oxidant-antioxidant balance might play a part in rendering the tissue more vulnerable to free radical induced injuries.

DNA Single Strand Breaks and Adenine Nucleotide Depletion as Indices of Oxidant Effects on Human Lung Cells

The comet assay (single cell gel electrophoresis) is a novel method to assess DNA strand breaks in single cells. We studied the oxidant sensitivity of cultured primary and transformed (MeT-5A) human pleural mesothelial cells, as well as primary and transformed (BEAS 2B) human bronchial epithelial cells, and compared the results obtained with the Comet assay to other markers of oxidant effects on cells, such as depletion of intracellular high-energy nucleotides (ATP, ADP, AMP), accumulation of products of nucleotide catabolism (xanthine, hypoxanthine, uric acid), and release of lactate dehydrogenase (LDH). The cells were exposed for 5 min to 4 h to 50–500 μM H 2O 2 or to 5–50 μM menadione. Significant tail moment increase, which is a marker of DNA strand breaks in the Comet assay, and intracellular nucleotide depletion occurred simultaneously in MeT-5A and BEAS 2B cells during the first 30–60 min of exposure to H 2O 2 and menadione. In the Comet assay variation between the individual cells could be detected. LDH release, a marker of cell injury, showed that mesothelial cells were far more sensitive than epithelial cells to oxidant-induced lytic cell injury. MeT-5A and BEAS 2B cells contained similar intracellular antioxidant enzyme activities, which may explain their similar oxidant sensitivity in the Comet assay. A significant increase (164%) in the tail moment was detectable in MeT-5A cells exposed to 50 μM H 2O 2 for 30 min. This returned to control level during the 4 h of continuing exposure. A 30 min exposure to 25 μM menadione caused a 61% increase in the mean tail moment but, unlike with H 2O 2, the change was irreversible during the following 4 h incubation. We conclude that human pleural mesothelial cells and bronchial epithelial cells show similar oxidant sensitivity when assessed by the Comet assay, but various oxidants differ in their potency in causing DNA breaks in these cells.

Effects of antioxidant enzyme modulations on interleukin-1-induced nuclear factor kappa B Activation

Nuclear factor kappa B (NF-κB) is a potent and pleiotropic transcription factor that can be activated by a wide variety of inducers, including interleukin-1 (IL-1). Although the detailed activation mechanism of NF-κB is still under investigation, it requires both phosphorylation and degradation of its inhibitory subunit Iκb and the presence of an oxidative environment. In this study, we systematically evaluated the influence of glutathione peroxidase, glutathione reductase and catalase on IL-1-induced NF-κB activation by analysing the effect of specific inhibitors of these enzymes. For the three antioxidant enzymes mentioned, their inhibition correlated with an overactivation of NF-κB, particularly for glutathione peroxidase. Inversely, we tested the response of glutathione peroxidase-transfected cells on NF-κB activation, which was lower as compared with the parental cells. Furthermore, interleukin-6 production also correlated perfectly with the reduced level of NF-κB activation in these experiments. The results clearly show that NF-κB activation is, strongly dependent on the antioxidant potential of the cells, especially on the activity of reduced glutathione-dependent enzymes such as glutathione peroxidase. The results support the hypothesis that the level of the oxidised glutathione:reduced glutathione ratio and the activity of intracellular antioxidant enzymes play a major role in NF-κB fine tuning.

Free-radical damage by UV or hypoxanthine-xanthine oxidase in cultured human skin fibroblasts: Protective effects of two human plasma fractions

Two in vitro methods to investigate free radical damage to cultured human skin fibroblasts have been used: irradiation with UVA or UVB, producing intracellular free radicals and DNA damage, or free radical production by the enzymatic system hypoxanthine-xanthine oxidase, releasing free radicals into the culture medium. These methods differ not only in the location of the free radicals generated but also in their nature and kinetics. The antioxidant properties of two human plasma extracts A and B (derived from Cohn's fraction IV and Cohn's fraction I + III) were investigated before, during and/or after the oxidative stress. Protection was observed when the fractions were added concomitantly with the enzymatic system (at 5 g/litre, fractions A and B exhibited, respectively, 77 and 50% activity) or during UVB irradiation (37 and 68% activity for fractions A and B, respectively at 5 g/litre). A small degree of protection was observed against UVA damage. No preventive or restorative effect was observed with the UVB system. The two fractions prevented UVA damage (at 2.5 g/litre, fraction A and B elicited 22 and 23% activity, respectively) but only fraction A also exhibited a restorative effect (at 5 g/litre, activity was 26%). One of the protective mechanisms could be the enhancement of intracellular antioxidant enzyme activity by incubation of cells with fractions A and B (after 24 hr of contact with fraction B, total glutathione peroxidase and glutathione peroxidase Se-dependent activities were significantly increased by 60 and 42%, respectively, compared with control values).

ADRIAMYCIN RESISTANCE IN CHINESE HAMSTER FIBROBLASTS FOLLOWING OXIDATIVE STRESS INDUCED BY PHOTODYNAMIC THERAPY

Photodynamic therapy (PDT) generates reactive oxygen species that are responsible for the initial cytotoxic events produced by this treatment. An extended (16 h) porphyrin incubation prior to light irradiation increased expression of the 75, 78 and 94 kDa glucose-regulated stress proteins (GRP), as well as the cognate form of the 70 kDa heat shock protein. However, these stress proteins were not induced following isoeffective PDT doses using a short (1 h) porphyrin incubation protocol. In the current study, Chinese hamster fibroblasts were used to examine sensitivity to adjunctive PDT and adriamycin as previous reports indicate a correlation between stress protein synthesis and a decrease in adriamycin cytotoxicity. Treatments that either induced GRP (i.e. PDT with an extended porphyrin incubation or exposure to the calcium ionophore A23187) or did not induce GRP (i.e. PDT with a short porphyrin incubation or UV irradiation) were followed at increasing time intervals with a 1 h adriamycin incubation. A time-dependent decrease in adriamycin cytotoxicity was observed when cells were first exposed to either of the PDT protocols or to A23187. Alterations in intracellular drug levels did not account for the change in adriamycin sensitivity. Likewise, intracellular glutathione concentrations and antioxidant enzyme activities were not significantly altered following PDT or A23187. Parameters associated with altered adriamycin sensitivity included a decrease in the percentage of S phase cells following PDT and A23187 as well as a depletion of intracellular ATP after PDT using the extended porphyrin incubation. These results demonstrate that PDT can be added to the growing list of diverse stresses producing transient resistance to adriamycin and that stress protein induction is not universally associated with all oxidative treatments inducing this resistance.

Pulmonary metabolism of reactive oxygen species.

Preexposure of rats to sublethal levels of hyperoxia or ozone reduces morbidity and mortality when the animals are subsequently exposed to lethal levels of either oxidant stress. Lung homogenates and isolated type II pneumocytes from rats exposed to these oxidant stresses demonstrate enhanced antioxidant enzyme activities. Antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase are responsible for the detoxification of partially reduced oxygen species, superoxide and hydrogen peroxide, to less reactive states. Potential pulmonary cellular loci of partially reduced oxygen include mitochondrial NADH dehydrogenase, endoplasmic reticulum-derived NADPH cytochrome c reductase, and cytosolic xanthine oxido reductase. Thus partially reduced oxygen species are hypothesized to mediate hyperoxia and ozone-induced pulmonary damage. This damage may be attenuated by enhanced intracellular antioxidant enzyme activities. Pharmacologic augmentation of pulmonary antioxidant enzymes may be accomplished via intratracheal or intravascular delivery of liposomes containing antioxidant enzymes. Rats pretreated with liposomes containing both superoxide dismutase and catalase, when subsequently exposed to lethal levels of hyperoxia, demonstrate enhanced survival compared with control animals or with animals treated with control liposomes or native antioxidant enzymes. Finally, knowledge obtained from in vitro investigations optimizing liposomal delivery to specific pulmonary cell types may further aid in reducing in vivo pulmonary damage to hyperoxia and ozone.