Overexpression of human glutathione peroxidase protects transgenic mice against focal cerebral ischemia/reperfusion damage

As stroke is a major cause of disability and death in the western world, there is great interest in the basic mechanisms by which ischemia/reperfusion (I/R) causes damage. To this end, extensive research has been carried out which identifies reactive oxygen species (ROS) as key participants in brain damage resultant from I/R. Brain tissue is protected from ROS damage by antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GP). Overexpression of SOD in transgenic mice has already been demonstrated to confer protection against I/R damage in murine stroke models. We are using transgenic mice overexpressing the intracellular form of glutathione peroxidase (GP1) to determine the protective capacity of overexpression of this enzyme on stroke damage. 1 h of focal cerebral ischemia followed by 24 h of reperfusion was induced using the intraliminal suture method. Volume of infarction was reduced by 48% in GP1 mice compared to nontransgenic littermates. Brain edema was reduced by 33%. Behavioral deficits agreed with histologic data. Overexpression of glutathione peroxidase confers significant protection against I/R damage in our stroke model possibly through direct scavenging of ROS or through the influencing of signalling mechanisms which lead to tissue damage.     

Satellite RNA is essential for encapsidation of groundnut rosette umbravirus RNA by groundnut rosette assistor luteovirus coat protein

This work deals with the antioxidant enzymatic response and the ultrastructural aspects of the skeletal muscle of young and aged rats kept under hypoxic or hyperoxic normobaric conditions. It is in fact well known that the supply of oxygen at concentrations higher or lower than those occurring under normal conditions can promote oxidative processes that can cause tissue damage. The enzymes investigated were both those directly involved in reactive oxygen species (ROS) scavenging (superoxide dismutase, catalase and selenium-dependent glutathione peroxidase), and those challenged with the detoxication of cytotoxic compounds produced by the action of ROS on biological molecules (glutathione transferase, glyoxalase I, glutathione reductase), in order to obtain a comparative view of the defence strategies used with respect to aging. Our results support the hypothesis that one of the major contributors to the aging process is the oxidative damage produced at least in part by an impairment of the antioxidant enzymatic system. This makes the aged organism particularly susceptible to oxidative stress injury and to the related degenerative diseases, especially in those tissues with high demand for oxidative metabolism.     

Alcohol-induced breast cancer: a proposed mechanism

Alcohol consumption increases the risk for breast cancer in women by still undefined means. Alcohol metabolism is known to produce reactive oxygen species (ROS), and breast cancer is associated with high levels of hydroxyl radical (*OH) modified DNA, point mutations, single strand nicks, and chromosome rearrangement. Furthermore, ROS modification of DNA can produce the mutations and DNA damage found in breast cancer. Alcohol dehydrogenase (ADH) and xanthine oxidoreductase (XOR) are expressed and regulated in breast tissues and aldehyde oxidase (AOX) may be present as well. Mammary gland XOR is an efficient source of ROS. Recently, hepatic XOR and AOX were found to generate ROS in two ways from alcohol metabolism: by acetaldehyde consumption and by the intrinsic NADH oxidase activity of both XOR and AOX. The data obtained suggests that: (1) expression of ADH and XOR or AOX in breast tissue provides the enzymes that generate ROS; (2) metabolism of alcohol produces acetaldehyde and NADH that can both be substrates for XOR or AOX and thereby result in ROS formation; and (3) ROS generated by XOR or AOX can induce the carcinogenic mutations and DNA damage found in breast cancer. Accumulation of iron coupled with diminished antioxidant defenses in breast tissue with advancing age provide additional support for this hypothesis because both result in elevated ROS damage that may exacerbate the risk for ROS-induced breast cancer.     

Oxygen free radicals and their clinical implications

Reactive oxygen species (ROS) have been implicated in a variety of pathological processes. The generation of highly reactive oxygen metabolites is an integral feature of normal cellular metabolism (mitochondrial respiratory chain, phagocytosis, arachidonic acid metabolism, ovulation and fertilization), however their production can multiply during pathological circumstances. Free oxygen radicals act either on the extracellular matrix or directly upon cellular membranes themselves. The fundamental defense of the organism against ROS include scavenger enzymes (superoxide dismutase, catalase, glutathione peroxidase) and lipid- and water soluble antioxidant compound (ascorbic acid, glutathione, albumin, transferrin, etc.). Their role in ischemia-reperfusion models have now been comprehensively investigated and it has become clear that ROS is to be blamed for the bulk of post-ischemic injuries, hence the basis for newly established antioxidant therapy in such cases. Also more and more studies have concluded a pivotal role of ROS in degenerative and inflammatory conditions, post-radiation processes and aging. Therefore it seems as we are continuously shedding light on the crucial part played by these molecules regarding a wide range of pathologies, we are discovering new therapeutic windows that would clinically assist us in managing such conditions.     

Oxidative stress in critical care: is antioxidant supplementation beneficial?

Reactive oxygen species (ROS) are constantly produced in human beings under normal circumstances. Antioxidant systems help defend the body against ROS but may be overwhelmed during periods of oxidative stress, which can cause lipid peroxidation, damage to DNA, and cell death. Critical illness, such as sepsis or adult respiratory distress syndrome, can drastically increase the production of ROS and lead to oxidative stress. Sources of oxidative stress during critical illness include activation of the phagocytic cells of the immune system (the respiratory burst), the production of nitric oxide by the vascular endothelium, the release of iron and copper ions and metalloproteins, and the vascular damage caused by ischemia reperfusion. Only indirect measurements of ROS are available, but the presence of oxidative stress in critical illness is supported by clinical studies. In general, serum antioxidant vitamin concentrations seem to decrease and measures of oxidative stress seem to increase in critically ill populations. Oxidative stress has been associated with sepsis, shock, a need for mechanical ventilation, organ dysfunction, acute respiratory distress syndrome, disseminated intravascular coagulation, surgery, and the presence of an acute-phase response. In addition, higher levels of oxidative stress seem to occur in patients with more notable injuries. Dietary supplementation with antioxidant vitamins seems to be the logical answer to decreasing serum antioxidant concentrations, but antioxidants may have adverse effects. The benefit of supplementing antioxidants in critically ill populations has not been shown and requires further study.     

Reactive oxygen species and antioxidants in signal transduction and gene expression

Reactive oxygen species (ROS) are produced by cellular metabolic reactions, and have been implicated in the pathogenesis of several diseases, including atherosclerosis, cancer, and Alzheimer's disease. Interestingly, clinical and epidemiologic studies have, in some cases, indicated that antioxidant nutrients may be effective in disease prevention. However, the efficacy of specific antioxidants in disease prevention is often both controversial and inconclusive. In an effort to elucidate the role of ROS and antioxidants in disease development and prevention, the chemistries of ROS and antioxidants have been examined extensively. Recently, molecular and cellular approaches have demonstrated that ROS and antioxidants can directly affect the cellular signaling apparatus and, consequently, the control of gene expression. This new research provides the link between ROS and antioxidant chemistries and the mechanisms of disease processes and prevention. This review illustrates how ROS function as potential intracellular and extracellular signaling molecules and how antioxidants can affect this process.     

A study and meta-analysis of lay attributions of cures for overcoming specific psychological problems

1. Lipoic acid is an example of an existing drug whose therapeutic effect has been related to its antioxidant activity. 2. Antioxidant activity is a relative concept: it depends on the kind of oxidative stress and the kind of oxidizable substrate (e.g., DNA, lipid, protein). 3. In vitro, the final antioxidant activity of lipoic acid is determined by its concentration and by its antioxidant properties. Four antioxidant properties of lipoic acid have been studied: its metal chelating capacity, its ability to scavenge reactive oxygen species (ROS), its ability to regenerate endogenous antioxidants and its ability to repair oxidative damage. 4. Dihydrolipoic acid (DHLA), formed by reduction of lipoic acid, has more antioxidant properties than does lipoic acid. Both DHLA and lipoic acid have metal-chelating capacity and scavenge ROS, whereas only DHLA is able to regenerate endogenous antioxidants and to repair oxidative damage. 5. As a metal chelator, lipoic acid was shown to provide antioxidant activity by chelating Fe2+ and Cu2+; DHLA can do so by chelating Cd2+. 6. As scavengers of ROS, lipoic acid and DHLA display antioxidant activity in most experiments, whereas, in particular cases, pro-oxidant activity has been observed. However, lipoic acid can act as an antioxidant against the pro-oxidant activity produced by DHLA. 7. DHLA has the capacity to regenerate the endogenous antioxidants vitamin E, vitamin C and glutathione. 8. DHLA can provide peptide methionine sulfoxide reductase with reducing equivalents. This enhances the repair of oxidatively damaged proteins such as alpha-1 antiprotease. 9. Through the lipoamide dehydrogenase-dependent reduction of lipoic acid, the cell can draw on its NADH pool for antioxidant activity additionally to its NADPH pool, which is usually consumed during oxidative stress. 10. Within drug-related antioxidant pharmacology, lipoic acid is a model compound that enhances understanding of the mode of action of antioxidants in drug therapy.     

High-affinity aptamers selectively inhibit human nonpancreatic secretory phospholipase A2 (hnps-PLA2)

Evidence is growing that reactive oxygen species (ROS), by-products of (normal) cellular aerobic metabolism, are involved in the pathogenesis of neurodegenerative diseases. One of these diseases is amyotrophic lateral sclerosis (ALS), in which motoneurons die, leading to paralysis and death. It remains uncertain whether ROS are the cause of (apoptotic) motoneuron death in ALS. To further understand the role of ROS in motoneuron death, we investigated the effects of ROS on isolated spinal rat motoneurons in culture. ROS were generated with a combination of iron(III) and ascorbate, or with hydrogen peroxide. Both toxic treatments resulted in a dose-dependent motoneuron death. Iron(III)/ascorbate toxicity was completely prevented with the hydrogen peroxide detoxifying enzyme catalase and partially prevented with the antioxidant vitamin E. SOD1, the enzyme that removes superoxide, did not protect against iron(III)/ascorbate toxicity. ROS treatment caused apoptotic motoneuron death: low doses of iron(III)/ ascorbate or hydrogen peroxide resulted in complete apoptosis ending in nuclear fragmentation, while high doses of ROS resulted in incomplete apoptosis (nuclear condensation). Thus, depending on the dose of ROS, the motoneurons complete the apoptotic pathway (low dose) or are stopped somewhere during this route (high dose).     

NHLBI workshop on the utilization of ECG databases: preservation and use of existing ECG databases and development of future resources

During the last years several reports have demonstrated that melatonin is a efficient free radical scavenger and general antioxidant. In addition, it has been shown that this neurohormone is able to increase the activity of glutathione peroxidase in rat brain cortex as well as the gene expression for some antioxidant enzymes in the Harderian gland of female Syrian hamster. Also, it is well known that brain cells are particularly exposed to free radicals, with antioxidant enzymes as the major defense mechanism that the brain uses to neutralize reactive oxygen species. The aim of the present study was to examine the influence of melatonin on gene expression for antioxidant enzymes in rat brain cortex. Our results clearly demonstrate that exogenously administered melatonin increases the levels of mRNA for glutathione peroxidase, copper-zinc superoxide dismutase, and manganese superoxide dismutase in this tissue. These stimulatory effects are observed after both acute and chronic treatment with this hormone, producing in the latter case the more marked increase. We therefore conclude that melatonin exerts an important role in providing indirect protection against free radical injury by stimulating gene expression for antioxidant enzymes. Consequently, melatonin could be considered as a potential therapeutic agent in some age-related neurodegenerative diseases where excessive free radical production has been implicated.     

Reduction of thymine hydroperoxide by phospholipid hydroperoxide glutathione peroxidase and glutathione transferases

Thymine hydroperoxide (5-hydroperoxymethyluracil), a model compound representing products of oxidative damage to DNA, is a substrate for glutathione peroxidase and some isoforms of glutathione transferase. In this paper, we show that selenium-dependent human phospholipid hydroperoxide glutathione peroxidase (Se-PHGPx) exhibits about four orders of magnitude higher activity on thymine hydroperoxide than that of other human enzymes such as selenium-dependent glutathione peroxidase and various representatives of glutathione transferases. The results indicate that Se-PHGPx may be an important enzyme in repairing oxidatively damaged DNA.     

Desferrioxamine and vitamin E protect against iron and MPTP-induced neurodegeneration in mice

To elucidate the neuroprotective effects of the iron chelator desferrioxamine (DFO) and the antioxidant vitamin E on excessive iron-induced free radical damage, a chronic iron-loaded mice model was established. The relationship between striatal iron content, oxidized to reduced glutathione ratio, hydroxyl radical (.OH) levels and dopamine concentrations were observed in DFO or vitamin E pretreated iron-loaded/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated C57BL/6 mice. The results demonstrated that both DFO and vitamin E inhibit the iron accumulation and thus reverses the increase in oxidized glutathione (GSSG), oxidized to reduced glutathione ratios, .OH and lipid peroxidation levels. The striatal dopamine concentration was elevated to normal value. Our data suggested that: (1) iron may induce neuronal damage and thus excessive iron in the brain may contribute to the neuronal loss in PD; (2) iron chelators and antioxidants may serve as potential therapeutic agents in retarding the progression of neurodegeneration.     

Detection of pro- and anti-inflammatory cytokines in stools of patients with inflammatory bowel disease

Astroglial cells protect neurons against oxidative damage. The antioxidant glutathione plays a pivotal role in the neuroprotective action of astroglial cells which is impaired following loss of glutathione. Anethole dithiolethione (ADT), a sulfur-containing compound which is used in humans as a secretagogue, increases glutathione levels in cultured astroglial cells under "physiological" conditions and is thought thereby to protect against oxidative damage. Presently, we report the effect of ADT (3-100 microM) on glutathione content of and efflux from rat primary astroglia-rich cultures under "pathological" conditions, i.e., extended deprivation of glucose and amino acids. Although cellular viability was not affected significantly, starvation of these cultures for 24 h in a bicarbonate buffer lacking glucose and amino acids led to a decrease in glutathione and protein content of approximately 43% and 40%, respectively. Although no effect on the protein loss occurred, the presence of ADT during starvation counteracted the starvation-induced loss of intracellular glutathione in a concentration-dependent way. At a concentration of 100 microM ADT even a significant increase in astroglial glutathione content was noted after 24 h of starvation. Alike intracellular glutathione levels, the amount of glutathione found in the buffer was elevated substantially if ADT was present during starvation. This ADT-mediated, apparent increase in glutathione efflux was additive to the stimulatory effect on extracellular glutathione levels of acivicin (100 microM), an inhibitor of extracellular enzymatic glutathione breakdown. However, the ADT-induced elevation of both intra- and extracellular glutathione content during starvation was prevented completely by coincubation with buthionine sulfoximine (10 microM), an inhibitor of glutathione synthesis. These results demonstrate that, most likely through stimulation of glutathione synthesis, ADT enables astroglial cells to maintain higher intra- and extracellular levels of glutathione under adverse conditions. Considering the lowered glutathione levels in neurodegenerative syndromes, we conclude that further evaluation of the therapeutic potential of the compound is warranted.     

Renal clearance, tissue distribution, and CA-125 responses in a phase I trial of suramin

Exposure of human spermatozoa to nicotinamide adenine dinucleotide phosphate (NADPH) resulted in the dose dependent generation of reactive oxygen species (ROS) which, at a critical level of intensity, induced lipid peroxidation, DNA damage and a dramatic decline of sperm motility. This system was then used as a model for screening the ability of different antioxidants to combat oxidative stress created through the excessive intracellular generation of toxic oxygen products of metabolism. A variety of antioxidants that has previously been shown to be protective against extracellularly derived oxidants (e.g. superoxide dismutase, catalase, vitamin E, hypotaurine) were ineffective in this system. Albumin, however, could provide complete protection against NADPH induced oxidative stress via mechanisms that did not involve the suppression of the lipid peroxidation cascade but rather the inactivation of lipid peroxides generated during this process. Albumin did not protect against DNA damage induced by NADPH but was extremely effective at preventing DNA fragmentation arising from the suppression of glutathione peroxidase activity with mercaptosuccinate. These studies emphasize that the design of clinically effective antioxidant treatments will depend, critically, upon the source of the oxidative stress. For cases involving excessive intracellular ROS generation, albumin appears to be an important means of neutralizing lipid peroxide-mediated damage to the sperm plasma membrane and DNA.     

Antioxidants in peripheral nerve

Oxidative stress and antioxidants have been related in a wide variety of ways with nervous tissue. This review attempts to gather the most relevant information related to a) the antioxidant status in non pathologic nervous tissue; b) the hypothesis and evidence for oxidative stress (considered as the disequilibrium between prooxidants and antioxidants in the cell) as the responsible mechanism of diverse neurological diseases; and c) the correlation between antioxidant alterations and neural function, in different experimental neuropathies. Decreased antioxidant availability has been observed in different neurological disorders in the central nervous system, for example, Parkinson's disease, Alzheimer's disease, epilepsy, amyotrophic lateral sclerosis, cerebral ischaemia, etc. Moreover, the experimental manipulation of the antioxidant defense has led in some cases to interesting experimental models in which electrophysiological alterations are associated with the metabolic modifications induced. In view of the electrophysiological and biochemical effects of some protein kinase C inhibitors on different neural experimental models, special attention is dedicated to the role of this kinase in peripheral nervous tissue. The nervous tissue, central as well as peripheral, has two main special features that are certainly related to its antioxidant metabolism: the lipid-enriched membrane and myelin sheaths, and cellular excitability. The former explains the importance of the glutathione (GSH)-conjugating activity towards 4-hydroxy-nonenal, a biologically active product of lipid peroxidation, present in nervous tissue and in charge of its inactivation. The impairment of the latter by oxidative damage or experimental manipulation of antioxidant metabolism is discussed. Work on different experimental neuropathies from author's laboratory has been primarily used to provide information about the involvement of free radical damage and antioxidants in peripheral nerve metabolic and functional impairment.     

Doppler ultrasound of blood flow velocities in ophthalmic and central retinal arteries during the early neonatal period

Dopamine has been implicated as a potential mediating factor in a variety of neurodegenerative disorders. Dopamine can be oxidized to form a reactive dopamine quinone that can covalently modify cellular macromolecules including protein and DNA. This oxidation can be enhanced through various enzymes including tyrosinase and/or prostaglandin H synthase. One of the potential targets in brain for dopamine quinone damage is tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. The present studies demonstrated that dopamine quinone, the formation of which was enhanced through the activity of the melanin biosynthetic enzyme, tyrosinase, covalently modified and inactivated tyrosine hydroxylase. Dihydroxyphenylalanine (DOPA; the catechol-containing precursor of dopamine) also inactivated tyrosine hydroxylase under these conditions. Catecholamine-mediated inactivation occurred with both purified tyrosine hydroxylase as well as enzyme present in crude pheochromocytoma homogenates. Inactivation was associated with covalent incorporation of radiolabelled dopamine into the enzyme as assessed by immunoprecipitation, size exclusion chromatography, and denaturing sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. Furthermore, the covalent modification and inactivation of tyrosine hydroxylase was blocked by antioxidant compounds (dithiothreitol, reduced glutathione, or NADH). In addition to kinetic feedback inhibition and the formation of an inhibitory dopamine/Fe+3 complex, these findings suggest that a third mechanism exists by which dopamine (or DOPA) can inhibit tyrosine hydroxylase, adding further complexity to the regulation of catecholamine biosynthesis.     

Central infusion of glucagon-like peptide-1-(7-36) amide (GLP-1) receptor antagonist attenuates lithium chloride-induced c-Fos induction in rat brainstem

Studies of neuronal injury and death after cerebral ischemia and various neurodegenerative diseases have increasingly focused on the interactions between mitochondrial function, reactive oxygen species (ROS) production and glutamate neurotoxicity. Recent findings suggest that increased mitochondrial ROS production precedes neuronal death after glutamate treatment. It is hypothesized that under pathological conditions when mitochondrial function is compromised, extracellular glutamate may exacerbate neuronal injury. In the present study, we focus on the relationship between mitochondrial superoxide production and glutamate neurotoxicity in cultured cortical neurons with normal or reduced levels of manganese-superoxide dismutase (MnSOD) activity. Our results demonstrate that neurons with reduced MnSOD activity are significantly more sensitive to transient exposure to extracellular glutamate. The increased sensitivity of cultured cortical neurons with reduced MnSOD activity is characteristically subject only to treatment by glutamate but not to other glutamate receptor agonists, such as N-methyl-d-aspartate, kainate and quisqualate. We suggest that the reduced MnSOD activity in neurons may exacerbate glutamate neurotoxicity via a mechanism independent of receptor activation.     

Studies on hepatic injury and antioxidant enzyme activities in rat subcellular organelles following in vivo ischemia and reperfusion

The activities of rat hepatic subcellular antioxidant enzymes were studied during hepatic ischemia/reperfusion. Ischemia was induced for 30 min (reversible ischemia) or 60 min (irreversible ischemia). Ischemia was followed by 2 or 24 h of reperfusion. Hepatocyte peroxisomal catalase enzyme activity decreased during 60 min of ischemia and declined further during reperfusion. Peroxisomes of normal density (d = 1.225 gram/ml) were observed in control tissues. However, 60 min of ischemia also produced a second peak of catalase specific activity in subcellular fractions corresponding to newly formed low density immature peroxisomes (d = 1.12 gram/ml). The second peak was also detectable after 30 min of ischemia followed by reperfusion for 2 or 24 h. Mitochondrial and microsomal fractions responded differently. MnSOD activity in mitochondria and microsomal fractions increased significantly (p < 0.05) after 30 min of ischemia, but decreased below control values following 60 min of ischemia and remained lower during reperfusion at 2 and 24 h in both organelle fractions. Conversely, mitochondrial and microsomal glutathione peroxidase (GPx) activity increased significantly (p < 0.001) after 60 min of ischemia and was sustained during 24 h of reperfusion. In the cytosolic fraction, a significant increase in CuZnSOD activity was noted following reperfusion in animals subjected to 30 min of ischemia, but 60 min of ischemia and 24 h of reperfusion resulted in decreased CuZnSOD activity. These studies suggest that the antioxidant enzymes of various subcellular compartments respond to ischemia/reperfusion in an organelle or compartment specific manner and that the regulation of antioxidant enzyme activity in peroxisomes may differ from that in mitochondria and microsomes. The compartmentalized changes in hepatic antioxidant enzyme activity may be crucial determinant of cell survival and function during ischemia/reperfusion. Finally, a progressive decline in the level of hepatic reduced glutathione (GSH) and concomitant increase in serum glutamate pyruvate transaminase (SGPT) activity also suggest that greater tissue damage and impairment of intracellular antioxidant activity occur with longer ischemia periods, and during reperfusion.     

Increased oxidative stress in rat liver and pancreas during progression of streptozotocin-induced diabetes

1. Oxygen free radicals have been suggested to be a contributory factor in complications of diabetes mellitus. There are many reports indicating the changes in parameters of oxidative stress in diabetes mellitus. In this study we aimed to identify whether oxidative stress occurs in the liver and pancreas in the initial stages of development of diabetes. 2. We therefore investigated the lipid peroxide level (thiobarbituric acid-reactive substances, TBARS) and activities of antioxidant enzymes [superoxide dismutase (SOD), catalase and glutathione peroxidase] in liver and pancreas of control and streptozotocin-induced diabetic rats at various stages of development of diabetes. 3. Male Sprague-Dawley rats were divided into two groups: group I, control (n = 42) and group II, diabetic (n = 42). Each group was further subdivided into seven groups consisting of six rats each. Rats in these subgroups were studied at weekly intervals (0 to 6 weeks). Plasma glucose levels, TBARS levels and activities of antioxidant enzymes were measured in liver and pancreas at various time intervals. 4. There was a significant (P < 0.05) and progressive increase in TBARS levels of liver and pancreas in the diabetic group. Total SOD and Cu-Zn-SOD activity increased (P < 0.05) with progression of diabetes while Mn-SOD activity showed no significant change in either tissue. Catalase and glutathione peroxidase activities increased significantly (P < 0.05) in liver and pancreas. 5. Immunohistochemical study of pancreatic islet revealed a decrease in the expression of insulin with progression of diabetes. However, glucagon and somatostatin showed an increase in immunoreactivity and a difference in their distribution pattern. 6. The findings of the present study suggest that oxidative stress starts at early onset of diabetes mellitus and increases progressively. In conclusion, the structural damage to these tissues or complications of diabetes mellitus may be due to oxidative stress.     

Changes in rat alveolar macrophageal antioxidant defense and reactive oxygen species release by high dietary vitamin E

For the past decade there has been emphasis on the immunomodulatory effects of vitamin E apart from its established role as a free radical scavenger. In alveolar macrophages (AMs), the role of vitamin E supplementation has not yet been investigated sufficiently. In the present study we have evaluated the effects of high vitamin E (DL-alpha-tocopheryl acetate, alpha-TA) supplementation for 10 d on rat-alveolar macrophageal antioxidant defense and reactive oxygen species (ROS) release. There was an increase in plasma vitamin E content from 5.22 +/- 1.30, at 50 mg to 12.23 +/- 1.06 and 22.32 +/- 2.02 micrograms/mL at 250 and 1,250 mg alpha-TA/kg dietary supplementation. Alveolar macrophage-vitamin E content enhanced by 56 to 75% at 250 and 1,250 mg alpha-TA diet as compared with control diet. Superoxide dismutase activity decreased and catalase and glutathione peroxidase activities increased significantly in AMs of 250 and 1,250 mg alpha-TA diet-fed rats. Reduced glutathione, total glutathione, and glutathione-S-transferase activity in AMs did not change significantly at any of the high doses of alpha-TA supplementation. On stimulation of AMs by phorbol-12-myristate-13-acetate (PMA), there was 2.8- and 3.5-fold enhancement in superoxide (O2-.) and H2O2 production, respectively, at 50 mg alpha-TA dose. But this increase by PMA could not take place in AMs from animals supplemented with 250 and 1,250 mg alpha-TA. It may therefore be concluded that high alpha-TA supplementation in diet may equip the AMs with a stronger defense against oxygen-free radical mediated damage to them.