Formation of acrolein-derived 2'-deoxyadenosine adduct in an iron-induced carcinogenesis model

Acrolein is a representative carcinogenic aldehyde found ubiquitously in the environment and formed endogenously through oxidation reactions, such as lipid peroxidation and myeloperoxidase-catalyzed amino acid oxidation. It shows facile reactivity toward DNA to form an exocyclic DNA adduct. To verify the formation of acrolein-derived DNA adduct under oxidative stress in vivo, we raised a novel monoclonal antibody (mAb21) against the acrolein-modified DNA and found that the antibody most significantly recognized an acrolein-modified 2' -deoxyadenosine. On the basis of chemical and spectroscopic evidence, the major antigenic product of mAb21 was the 1,N6-propano-2' -deoxyadenosine adduct. The exposure of rat liver epithelial RL34 cells to acrolein resulted in a significant accumulation of the acrolein-2' -deoxyadenosine adduct in the nuclei. Formation of this adduct under oxidative stress in vivo was immunohistochemically examined in rats exposed to ferric nitrilotriacetate, a carcinogenic iron chelate that specifically induces oxidative stress in the kidneys of rodents. It was observed that the acrolein-2' -deoxyadenosine adduct was formed in the nuclei of the proximal tubular cells, the target cells of this carcinogenesis model. The same cells were stained with a monoclonal antibody 5F6 that recognizes an acrolein-lysine adduct, by which cytosolic accumulation of acrolein-modified proteins appeared. Similar results were also obtained from myeloperoxidase knockout mice exposed to the iron complex, suggesting that the myeloperoxidase-catalyzed oxidation system might not be essential for the generation of acrolein in this experimental animal carcinogenesis model. The data obtained in this study suggest that the formation of a carcinogenic aldehyde through lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress.     

Effects of new antioxidant compounds PNU-104067F and PNU-74389G on antioxidant defense in normal and diabetic rats

Diabetes mellitus and its complications are associated with elevated oxidative stress, leading to much interest in antioxidant compounds as possible therapeutic agents. Two new classes of antioxidant compounds, the pyrrolopyrimidines and the 21-aminosteroids, are known to inhibit lipid peroxidation and other biomolecular oxidation. We hypothesized that in the presence of excess oxidants or the impaired antioxidant defense seen in diabetes mellitus, administration of antioxidants such as these may reverse the effects of diabetes on antioxidant parameters. This study measured the effects of subchronic (14 day) treatment with a pyrrolopyrimidine (PNU-104067F) or a 21-aminosteroid (PNU-74389G) in normal and diabetic Sprague-Dawley rats. Activity levels of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, concentrations of oxidized and reduced glutathione, and lipid peroxidation were used as measures of antioxidant defense in liver, kidney, heart, and brain tissue. In normal rats, the only effect was a 43% increase in cardiac lipid peroxidation after treatment with PNU-104067F. In diabetic rats, the only reversals of the effects of diabetes were a 30% decrease in hepatic glutathione peroxidase activity after PNU-74389G treatment and a 33% increase in cardiac glutathione disulfide concentration after PNU-104067F treatment. In contrast to these effects, increased cardiac glutathione peroxidase and catalase activities, increased brain glutathione peroxidase activity, increased hepatic lipid peroxidation, decreased hepatic glutathione content, and decreased hepatic catalase activity were seen in diabetic rats, reflecting an exacerbation of the effects of diabetes.     

Aldo-keto reductase 1B7 is a target gene of FXR and regulates lipid and glucose homeostasis

Aldo-keto reductase 1B7 (AKR1B7) is proposed to play a role in detoxification of by-products of lipid peroxidation. In this article, we show that activation of the nuclear receptor farnesoid X receptor (FXR) induces AKR1B7 expression in the liver and intestine, and reduces the levels of malondialdehyde (MDA), the end product of lipid peroxidation, in the intestine but not in the liver. To determine whether AKR1B7 regulates MDA levels in vivo, we overexpressed AKR1B7 in the liver. Overexpression of AKR1B7 in the liver had no effect on hepatic or plasma MDA levels. Interestingly, hepatic expression of AKR1B7 significantly lowered plasma glucose levels in both wild-type and diabetic db/db mice, which was associated with reduced hepatic gluconeogenesis. Hepatic expression of AKR1B7 also significantly lowered hepatic triglyceride and cholesterol levels in db/db mice. These data reveal a novel function for AKR1B7 in lipid and glucose metabolism and suggest that AKR1B7 may not play a role in detoxification of lipid peroxides in the liver. AKR1B7 may be a therapeutic target for treatment of fatty liver disease associated with diabetes mellitus.     

Effects of Oxidative Stress Caused by Hyperoxia and Diquat. A Study in Isolated Hepatocytes

The effects of oxidative stress caused by hyperoxia or administration of the redox active compound diquat were studied in isolated hepatocytes, and the relative contribution of lipid peroxidation, glutathione (GSH) depletion, and NADPH oxidation to the cytotoxicity of active oxygen species was investigated.

The redox cycling of diquat occurred primarily in the microsomal fraction since diquat was found not ' to penetrate into the mitochondria. Depletion of intracellular GSH by pretreatment of the animals with diethyl maleate promoted lipid peroxidation and sensitized the cells to oxidative stress. Diquat toxicity was also greatly enhanced when glutathione reductase was inhibited by pretreatment of the cells with 1,3-bis(2-chloroethyI)-1-nitrosourea. Despite extensive lipid peroxidation, loss of cell viability was not observed, with either hyperoxia or diquat, until the GSH level had fallen below ≈ 6 nmol/10⁶ cells.

The iron chelator desferrioxamine provided complete protection against both diquat-induced lipid peroxidation and loss of cell viability. In contrast, the antioxidant a-tocopherol inhibited lipid peroxidation but provided only partial protection from toxicity. The hydroxy! radical scavenger α-keto-γ-methiol butyric acid, finally, also provided partial protection against diquat toxicity but had no effect on lipid peroxidation.

The results indicate that there is a critical GSH level above which cell death due to oxidative stress is not observed. As long as the glutathione peroxidase – glutathione reductase system is unaffected, even relatively low amounts of GSH can protect the cells by supporting glutathione peroxidase-mediated metabolism of H₂O₂ and lipid hydroperoxides.     

Immunohistochemical study of glutathione reductase in rat ocular tissues at different developmental stages

Glutathione, which is found in high levels in eye tissues, is involved in multiple functions, including serving as an antioxidant and as an electron donor for peroxidases. Although the activities of enzymes related to glutathione metabolism have been reported in the eye, the issue of which cells produce these proteins, where they are produced and at what levels is an important one. Glutathione reductase, an enzyme which recycles oxidized glutathione by transferring electrons from NADPH, was localized immunohistochemically in adult rat eye in this study. The reductase was distributed in the corneal and conjunctival epithelia, corneal keratocytes and endothelium, iridial and ciliary epithelia, neural retina, and retinal pigment epithelium. In addition, it was highly expressed in ganglion cells, which are responsible for transmitting photophysiological signals from the retina to the higher visual centres. To clarify the correlation of glutathione reductase expression and oxidative stress, the enzymatic activity and the level of protein expression at the pre- and postnatal stages was examined. Expression of the enzyme was detected first in the ganglion cell layer of a late prenatal stage, and appeared in the inner plexyform layer after birth. Along with an increasing differentiation between the inner nuclear and outer nuclear layers, glutathione reductase expression became detectable in the outer plexyform layer. Pigment epithelial cells were positively stained only after birth. Expression was also detected in the lens epithelium from the prenatal to early postnatal stages although its level was low in the adult lens. Collectively, these data, except for lens epithelia, suggest the pivotal role of glutathione reductase in recycling oxidized glutathione for the protection of the tissues against oxidative stress, which is caused by eye opening accompanied by the initiation of various ocular processes, such as accession of light and transduction of the photochemical signal.     

The effect of 2,4-dichlorophenol and pentachlorophenol on antioxidant system in the leaves of Phalaris arudinacea

The purpose of this work was to evaluate the effect of 2,4-dichlorophenol (2,4-DCP) and pentachlorophenol (PCP) on the activity of antioxidative system and lipid peroxidation in the leaves of reed canary grass (Phalaris arudinacea). The activity of catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR) and glutathione S-transferase (GST) as well as the content of glutathione, ascorbate and phenolic compounds were determined. An induced-increase in the APX, CAT, GPX and GR activities was stronger for PCP, while a significant increase in the GST activity was noted only for 2,4-DCP. Both compounds increased the content of phenolic compounds, oxidized and reduced glutathione as well as the content of ascorbic acid. PCP induced stronger increase in lipid peroxidation than 2,4-DCP. The observed changes revealed that chlorophenols induce oxidative stress and oxidative damage in the leaves of reed canary grass.     

Colocalization of polyol-metabolizing enzymes and immunological detection of fructated proteins in the female reproductive system of the rat

The expression of aldose reductase (AR) and sorbitol dehydrogenase (SDH), which, in concert, catalyze the conversion of glucose to fructose via sorbitol, in the rat ovary, oviduct, and uterus, was investigated by immunohistochemical and biochemical analyses. The activities and protein levels of AR and SDH were higher in the ovary than in the oviduct and uterus. A strong immunoreactivity to the anti-AR antibody was observed in granulosa cells and epithelia of the oviduct, endometrium, and endometrial glands, and virtually the same tissues were strongly stained with the anti-SDH antibody. The application of an anti-fructated lysine antibody, which detects an adduct of fructose with the epsilon-amino group of lysine in proteins, in this study detected marked staining mainly in the egg and luminal surface of the oviductal epithelia. Collectively, these data indicate that fructose is produced by coordinately expressed AR and SDH in the egg and epithelia of the oviduct and suggest that the resulting sorbitol and fructose can be used as energy sources for spermatozoa motility during the fertilization process. The abundance of AR compared with SDH suggests that it also plays an additional role in the reproductive system, which might include a source of reducing power and protection against toxic carbonyl compounds.     

Functional significance of the pentose phosphate pathway and glutathione reductase in the antioxidant defenses of human sperm

Glutathione peroxidase is one of the principal antioxidant defense enzymes in human spermatozoa, but it requires oxidized glutathione to be reduced by glutathione reductase using NADPH generated in the pentose phosphate pathway. We investigated whether flux through the pentose phosphate pathway would increase in response to oxidative stress and whether glutathione reductase was required to protect sperm from oxidative damage. Isotopic measurements of the pentose phosphate pathway and glycolytic flux, thiobarbituric acid assay of malondialdehyde for lipid peroxidation, and computer-assisted sperm analysis for sperm motility were assessed in a group of normal, healthy semen donors. Applying moderate oxidative stress to human spermatozoa by adding cumene hydroperoxide, H(2)O(2), or xanthine plus xanthine oxidase or by promoting lipid peroxidation with ascorbate increased flux through the pentose phosphate pathway without changing the glycolytic rate. However, adding higher concentrations of oxidants inhibited both the pentose phosphate pathway and glycolytic flux. At concentrations of 50 microg/ml or greater, the glutathione reductase-inhibitor 1,3-bis-(2-chloroethyl) 1-nitrosourea decreased flux through the pentose phosphate pathway and blocked the response to cumene hydroperoxide. It also increased lipid peroxidation and impaired the survival of motility in sperm incubated under 95% O(2). These data show that the pentose phosphate pathway in human spermatozoa can respond dynamically to oxidative stress and that inhibiting glutathione reductase impairs the ability of sperm to resist lipid peroxidation. We conclude that the glutathione peroxidase-glutathione reductase-pentose phosphate pathway system is functional and provides an effective antioxidant defense in normal human spermatozoa.     

Impact of chromium on lipoperoxidative processes and subsequent operation of the glutathione cycle in rat renal system.

Oral administration of K2Cr2O7 to male albino rats at an acute dose of 1500 mg/kg body wt/day for 3 days brought about sharp decrease in the activities of glucose-6-phosphate dehydrogenase and glutathione reductase of kidney epithelial cells. The scavenging system of kidney epithelium is also affected as evident by the highly significant fall in the activities of glutathione peroxidase, superoxide dismutase and catalase which ultimately leads to the increase in lipid peroxidation value in kidney cortical homogenate. However, glutathione-s-transferase activity in cytosol and glutathione and total thiol content in cortical homogenate were not altered. Chronic oral administration of K2Cr2O7 (300 mg/kg body wt/day) for 30 days to rats lead to elevation in the activities of glutathione peroxidase, glutathione reductase, glutathione-s-transferase, superoxide dismutase and catalase with no change in glucose-6-phosphate dehydrogenase activity in epithelial cells. This might lead to the increase in glutathione and total thiol status and decrease in lipid peroxidation value in whole homogenate system.     

High-resolution crystal structure of AKR11C1 from Bacillus halodurans: an NADPH-dependent 4-hydroxy-2,3-trans-nonenal reductase

Aldo-keto reductase AKR11C1 from Bacillus halodurans, a new member of aldo-keto reductase (AKR) family 11, has been characterized structurally and biochemically. The structures of the apo and NADPH bound form of AKR11C1 have been solved to 1.25 A and 1.3 A resolution, respectively. AKR11C1 possesses a novel non-aromatic stacking interaction of an arginine residue with the cofactor, which may favor release of the oxidized cofactor. Our biochemical studies have revealed an NADPH-dependent activity of AKR11C1 with 4-hydroxy-2,3-trans-nonenal (HNE). HNE is a cytotoxic lipid peroxidation product, and detoxification in alkaliphilic bacteria, such as B.halodurans, plays a crucial role in survival. AKR11C1 could thus be part of the detoxification system, which ensures the well being of the microorganism. The very poor activity of AKR11C1 on standard, small substrates such as benzaldehyde or DL-glyeraldehyde is consistent with the observed, very open active site lacking a binding pocket for these substrates. In contrast, modeling of HNE with its aldehyde function suitably positioned in the active site suggests that its elongated hydrophobic tail occupies a groove defined by hydrophobic side-chains. Multiple sequence alignment of AKR11C1 with the highly homologous iolS and YqkF proteins shows a high level of conservation in this putative substrate-binding site. We suggest that AKR11C1 is the first structurally characterized member of a new class of AKRs with specificity for substrates with long aliphatic tails.     

Antioxidant status,lipid peroxidation,mixed function oxidase and UDP-glucuronyl transferase activities in livers from control and DOCA salt-hypertensive male Sprague Dawley rats

The effects of DOCA-salt hypertensive treatment on hepatic glutathione-dependent defense system, antioxidant enzymes, lipid peroxidation, mixed function oxidase and UDP-glucuronyl transferase activities were investigated in male Sprague Dawley rats.Compared with controls, DOCA-salt hypertensive rats had lower body weights (linked to liver hypertrophy). Mixed function oxidase and p-nitrophenol-UGT activities were not affected by the treatment but a significant lower rate of the glucuronoconjugation rate of bilirubin (p < 0.001) was observed in DOCA-salt hypertensive rats. While cytosolic glutathione contents and glutathione reductase activity were not affected, glutathione peroxidase (p < 0.001), glutathione transferase (p < 0.001) and catalase (p < 0.01) activities were decreased and associated with higher malondialdehyde contents (p < 0.001) in treated rats. The imbalance in liver antioxidant status (increasing generation of cellular radical species), associated with increases in lipid peroxidation, suggests that oxidative stress might be directly related to arterial hypertension in DOCA-salt treated male Sprague Dawley rats.     

Oxidative metabolism and protoplast culture

Summary Barley leaf blade protoplasts accumulate malonaldehyde, a product of lipid peroxidation, during culture. In addition, glutathione levels fall after protoplast isolation and the proportion of glutathione in the oxidized state rises. These data indicate oxidative stress after protoplast isolation and during culture. The cause of this phenomenon is revealed by data showing that the activities of enzymes associated with antioxidative processes including glutathione reductase and ascorbate peroxidase decrease after barley protoplast isolation. In contrast, protoplasts isolated from suspension cultured cells of bromegrass and soybean exhibit little evidence for oxidative stress and increased activities of glutathione reductase and ascorbate peroxidase. We suggest that an antioxidative response is associated with mitosis and colony formation from protoplasts, as exhibited by bromegrass and soybean. Conversely, failure of an antioxidative response is associated with low viability and absence of mitosis, as in barley. Increased viability of barley leaf protoplasts cultured on feeder layer cells is correlated with increased glutathione content and higher glutathione reductase activity.     

Effects of isoeugenol on oxidative stress pathways in normal and streptozotocin-induced diabetic rats.

Because some complications of diabetes mellitus may result from oxidative damage, we investigated the effects of subacute treatment (10mg/kg/day, intraperitoneal [ip], for 14 days) with the antioxidant isoeugenol on the oxidant defense system in normal and 30-day streptozotocin-induced diabetic Sprague-Dawley rats. Liver, kidney, brain, and heart were assayed for degree of lipid peroxidation, reduced and oxidized glutathione content, and activities of the free radical-detoxifying enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase. All tissues from diabetic animals exhibited disturbances in antioxidant defense when compared with normal controls. Treatment with isoeugenol reversed diabetic effects on hepatic glutathione peroxidase activity and on oxidized glutathione concentration in brain. Treatment with the lipophilic compound isoeugenol also decreased lipid peroxidation in both liver and heart of normal animals and decreased hepatic oxidized glutathione content in both normal and diabetic rats. Some effects of isoeugenol treatment, such as decreased activity of hepatic superoxide dismutase and glutathione reductase in diabetic rats, were unrelated to the oxidative effects of diabetes. In heart of diabetic animals, isoeugenol treatment resulted in an exacerbation of already elevated activities of catalase. These results indicate that isoeugenol therapy may not reverse diabetic oxidative stress in an overall sense.     

Effects of piperine on antioxidant pathways in tissues from normal and streptozotocin-induced diabetic rats

Using diabetes mellitus as a model of oxidative damage, this study investigated whether subacute treatment (10 mg/kg/day, intraperitoneally for 14 days) with the compound piperine would protect against diabetes-induced oxidative stress in 30-day streptozotocin-induced diabetic Sprague-Dawley rats. Liver, kidney, brain, and heart were assayed for degree of lipid peroxidation, reduced and oxidized glutathione (GSH and GSSG, respectively) content, and activities of the free-radical detoxifying enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase. Piperine treatment of normal rats enhanced hepatic GSSG concentration by 100% and decreased renal GSH concentration by 35% and renal glutathione reductase activity by 25% when compared to normal controls. All tissues from diabetic animals exhibited disturbances in antioxidant defense when compared with normal controls. Treatment with piperine reversed the diabetic effects on GSSG concentration in brain, on renal glutathione peroxidase and superoxide dismutase activities, and on cardiac glutathione reductase activity and lipid peroxidation. Piperine treatment did not reverse the effects of diabetes on hepatic GSH concentrations, lipid peroxidation, or glutathione peroxidase or catalase activities; on renal superoxide dismutase activity; or on cardiac glutathione peroxidase or catalase activities. These data indicate that subacute treatment with piperine for 14 days is only partially effective as an antioxidant therapy in diabetes.     

Effects of hyperglycemia on sperm and testicular cells of Goto-Kakizaki and streptozotocin-treated rat models for diabetes

Diabetes mellitus is a degenerative disease that has deleterious effects on male reproductive function, possibly through an increase in oxidative stress. This study was conducted in order to clarify the mechanisms by which oxidative stress influences animal models for both type 1 (streptozotocin-treated rats, STZ) and type 2 (Goto-Kakizaki (GK) rats) diabetes. We determined the extent of lipid peroxidation, protein oxidation, lactate levels, adenine nucleotides, adenylate energy charge and the activity of glutathione peroxidase, glutathione reductase and lactate dehydrogenase, in isolated testicular cells of control and diabetic rats. We have also correlated these parameters with sperm count and motility. Sperm concentration and motility were decreased in STZ-treated rats. ATP levels were lower in rats treated with STZ for 3 months, in contrast to GK and rats treated with STZ for 1 month, suggesting an adaptative response. STZ-treated rats showed increased lipid peroxidation after 1 week and 3 months of treatment. Glutathione reductase (G-red) activity was found to be higher in GK rats. Glutathione peroxidase activity was lower in GK and rats treated with STZ for 1 month, which is in accordance with the proposal of functional recovery in these animals. We conclude that hyperglycemia has an adverse effect in sperm concentration and motility via changes in energy production and free radical management. Furthermore, both animal models, particularly GK rats and rats treated with STZ for 1 month, present some metabolic adaptations, increasing the efficiency of mitochondrial ATP production, in order to circumvent the deleterious effects promoted by the disease.     

Antioxidant enzymatic systems in neuronal and glial cell-enriched fractions of rat brain during aging

The activities of Cu,Zn superoxide dismutase, glutathione peroxidase, catalase and glutathione reductase in neuronal and glial cell-enriched fractions obtained from the cerebral cortex of rat brain during aging (15, 30, 90, 350, 750 days of age) were assayed. Our results showed that glutathione peroxidase, catalase and glutathione reductase activities varied little during the examined periods. Only the Cu,Zn superoxide dismutase activity decreased notably from 15th to 750th day of age in both neuronal and glial cells, moreover the activities of all enzymes studied were always detected at lower levels in neuronal cells with respect to glial cells. In agreement with diminished SOD activity, the lipid peroxidation showed an elevated increase with aging; this fact is more evident in neuronal than in glial cells. In conclusion our data show that Cu,Zn superoxide dismutase is the most affected antioxidant enzymatic system of brain aging and it could be responsible for the increased lipid peroxidation in both cell types examined.A preliminary report of these results was presented at the 19th Meeting F.E.B.S. Rome July 2–7, 1989.     

Regulation of the aldo-keto reductase gene akr1b7 by the nuclear oxysterol receptor LXRalpha (liver X receptor-alpha) in the mouse intestine: putative role of LXRs in lipid detoxification processes

Liver X receptors (LXRs) regulate the expression of a number of genes involved in cholesterol and lipid metabolism after activation by their cognate oxysterol ligands. AKR1-B7 (aldo-keto reductase 1-B7) is expressed in LXR target tissues such as intestine, and because of its known role in detoxifying lipid peroxides, we investigated whether the AKR1-B7 detoxification pathway was regulated by LXRs. Here we show that synthetic LXR agonists increase the accumulation of AKR1-B7 mRNA and protein levels in mouse intestine in wild-type but not lxr(-/-) mice. Regulation of akr1b7 by retinoic X receptor/LXR heterodimers is dependent on three response elements in the proximal murine akr1b7 promoter. Two of these cis-acting elements are specific for regulation by the LXRalpha isoform. In addition, in duodenum of wild-type mice fed a synthetic LXR agonist, we observed an LXR-dependent decrease in lipid peroxidation. Our results demonstrate that akr1b7 is a direct target of LXRs throughout the small intestine, and that LXR activation plays a protective role by decreasing the deleterious effects of lipid peroxides in duodenum. Taken together, these data suggest a new role for LXRs in lipid detoxification.     

Human aldo-keto reductase AKR7A2 protects against the cytotoxicity and mutagenicity of reactive aldehydes and lowers intracellular reactive oxygen species in hamster V79-4 cells

Aldo-keto reductase (AKR) enzymes are critical for the detoxication of endogenous and exogenous aldehydes. Previous studies have shown that the AKR7A2 enzyme is catalytically active toward aldehydes arising from lipid peroxidation, suggesting a potential role against the consequences of oxidative stress, and representing an important detoxication route in mammalian cells. The aim of this study was to determine the ability of AKR7A2 to protect cells against aldehyde cytotoxicity and genotoxicity and elucidate its potential role in providing resistance to oxidative stress. A transgenic mammalian cell model was developed in which AKR7A2 was overexpressed in V79-4 cells and used to evaluate the ability of AKR7A2 to provide resistance against toxic aldehydes. Results show that AKR7A2 provides increased resistance to the cytotoxicity of 4-hydroxynonenal (HNE) and modest resistance to the cytotoxicity of trans, trans-muconaldehyde (MUC) and methyglyoxal, but provided no protection against crotonaldehyde and acrolein. Cells expressing AKR7A2 were also found to be less susceptible to DNA damage, showing a decrease in mutation rate cause by 4-HNE compared to control cells. Furthermore, the role of the AKR7A2 enzyme on the cellular capability to cope with oxidative stress was assessed. V79 cells expressing AKR7A2 were more resistant to the redox-cycler menadione and were able to lower menadione-induced ROS levels in both a time and dose dependent manner. In addition, AKR7A2 was able to maintain intracellular GSH levels in the presence of menadione. Together these findings indicate that AKR7A2 is involved in cellular detoxication pathways and may play a defensive role against oxidative stress in vivo.     

Protective role of L-methionine against free radical damage of rat brain synaptosomes

Incubation of rat brain synaptosomal/mitochondrial fraction with tert-butylhydroperoxide resulted in accumulation of the lipid peroxidation product, conjugated dienes, damage of the synaptosomal membrane as evidenced by leakage of lactate dehydrogenase, and decrease of the total content of glutathione and of the GSH/GSSG ratio. This treatment also produced a considerable decrease of the ouabain-sensitive ATPase activity and a much smaller diminution of the activities of glutathione reductase and glutathione transferase. Preincubation of the synaptosomal/mitochondrial fraction with 0.5 or 1.0 mM L-methionine significantly protected against lipid peroxidation, membrane damage and changes in the glutathione system produced by low (1 mM) concentrations of tert-butylhydroperoxide and completely prevented inactivation of ouabain-sensitive ATPase, glutathione reductase and glutathione transferase by such treatment. The importance of L-methionine in antioxidant protection is discussed.